Optical switch protein

Abstract

The present invention addresses the problem of providing a photoswitch protein capable of effectively controlling binding and dissociation of a protein compared to conventional photoswitch proteins. The invention relates to an optical switch protein, which is different from the existing optical switch protein consisting of two independent proteins, and is an optical switch protein consisting of a polypeptide. More specifically, the switch protein according to the present invention is, for example, a protein in which the C-terminal of a protein (a) and the N-terminal of a protein (b) are directly or indirectly linked. (a) A protein comprising an amino acid sequence having 80% or more sequence homology with an amino acid sequence obtained by deleting X contiguous amino acid residues from the C-terminal residue to the N-terminal side of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 36, where X is an integer of 0-6. (b) a protein comprising an amino acid sequence having 80% or more sequence homology with an amino acid sequence obtained by deleting Y contiguous amino acid residues from the N-terminal residue to the C-terminal side of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 36, Y being an integer of 0-55.

Description

Technical Field

[0001] This invention relates to photoreceptor proteins (photoswitch proteins) that can control the binding and dissociation of interacting proteins. Background Technology

[0002] Proteins, as one of the building blocks of living organisms, perform various functions within the body. For example, they interact with each other to participate in the control of systems that form the basis of life, such as signal transduction and substance transport. In recent years, with the development of optogenetics, techniques for regulating protein-protein interactions within organisms through light manipulation have been developed; for example, photo-switching proteins are now attracting much attention. Initially, photo-switching proteins were reported as proteases whose binding and dissociation are controlled by the ON / OFF state of light irradiation (Patent Document 1). Various intracellular phenomena, such as signal transduction, genome editing, and gene expression, can be regulated by using light manipulation to control the binding and dissociation of photo-switching proteins (Non-Patent Document 1 and Non-Patent Document 2).

[0003] Vivid (VVD), a protein derived from *Neurospora Crassa*, rapidly forms homodimers upon receiving blue light (Non-Patent Literature 3). Composed of approximately 150 amino acids, VVD has a smaller molecular weight compared to previously reported photoreceptor-based dimerized proteins, facilitating precise molecular design. Furthermore, the cofactor required for VVD activity (flavin adenine dinucleotide) is present in eukaryotic cells, thus offering excellent utilization for functionalization in mammalian cells. However, VVD suffers from low binding affinity upon light irradiation and an inability to selectively bind to specific targets due to homodimer formation.

[0004] To address the aforementioned problems with VVD, the inventors discovered that modifying the α-helix amino acid on the N-terminal side of VVD allows for control of dimer formation efficiency, and modifying the amino acid adjacent to the flavin-binding domain allows for control of dimer dissociation rate. Furthermore, it was learned that by applying different modifications to the groups forming dimers—namely, modifications to the aforementioned amino acids of VVD—dimers can be formed between VVD proteins with different modifications (Patent Document 1, Non-Patent Document 1). These VVD proteins with different amino acid modifications are hereinafter referred to as "Magnets," and the control system for the protein using magnets is also referred to as a "magnet system."

[0005] The magnet system developed by the inventors comprises a VVD (also referred to as "positive magnet (pMag)") in which at least one of the amino acids in the contact region during dimer formation is replaced with a basic amino acid (an amino acid with a positive charge) and a VVD (also referred to as "negative magnet (nMag)") in which an acidic amino acid (an amino acid with a negative charge) is replaced. It is expected that the pMag and nMag group will serve as a light-switching protein to improve the aforementioned problems of wild-type VVD. However, the binding efficiency of the interacting proteins based on the two independent molecules of pMag and nMag depends on the frequency of molecular interaction, and when using a bimolecular type magnet system in cells, it depends on the intracellular concentration of the magnet protein. That is, although a magnet system exhibiting improved control efficiency is possible, there is still room for improvement in increasing the interaction frequency.

[0006] Existing technical documents

[0007] Patent documents

[0008] Patent Document 1: Japanese Patent Application Publication No. 2015-165776

[0009] Non-patent literature

[0010] Non-patent literature 1: Kawano et al., Nature Chemical Biology 12: 1059-1064 (2016).

[0011] Non-patent literature 2: Kawano et al., Nature Communications 6:6256 (2015).

[0012] Non-patent literature 3: Zoltowski et al., Science 316: 1054-1057 (2007).

[0013] Non-patent literature 4: Nihongaki et al., Nat. Biotechnol. 33:755-760 (2015). Summary of the Invention

[0014] In view of the above, the objective of the present invention is to provide a light-switching protein that can effectively control the binding and dissociation of proteins compared with existing light-switching proteins (such as magnets).

[0015] To address the aforementioned issues, the inventors have developed a single-molecule type of photoswitch protein formed by tandemly linking pMag and nMag, which constitute the aforementioned magnet. The results showed that, compared to existing bimolecular photoswitch proteins, it significantly improves the control efficiency of binding and dissociation of split proteins (protein fragments obtained by splitting a single protein in a manner capable of reassociation). Hereinafter, the single-molecule type of photoswitch protein formed by tandemly linking VVD proteins or their mutants (pMag, nMag, etc.) will also be referred to as "scMagnet".

[0016] The inventors designed scMagnet based on the three-dimensional structural models of pMag and nMag. When pMag and nMag are directly connected, if inference is made based on the structural models of pMag and nMag in the dark, it is inferred that the N-terminal fragments and C-terminal fragments of each segmented protein bound to pMag and nMag are at least 6 nm apart. Figure 1 (a). Therefore, it is believed that by tandemly linking pMag and nMag, the self-rebinding of segmentation proteins in the dark can be suppressed. Figure 1 (a). On the other hand, if we infer from the structural model of the VVD dimer (a dimer composed of pMag and nMag) under light irradiation, we infer that the N-terminus of pMag and the C-terminus of nMag are close to each other within a 1 nm range, which is considered to promote the rebinding of the split proteins. Figure 1 (b).

[0017] Based on the above research, the inventors believe that scMagnet undergoes a significant structural change from an open shape to a closed shape in a light-dependent manner. By utilizing the intermolecular interactions between pMag and nMag under light irradiation, the binding and dissociation of proteins (e.g., fragmented protein segments) bound to pMag and nMag can be controlled very effectively. Figure 1 c).

[0018] In fact, the inventors investigated the usefulness of scMagnet using a split-activation Cas9 endonuclease (PA-Cas9) (Non-Patent Document 4) under experimental conditions where existing bimolecular photoswitch systems could not operate well (e.g., low expression levels of photoswitch proteins). The results showed that scPA-Cas9, as a single-molecule photoswitch protein, exhibited more than 10 times the photoinduction efficiency under light irradiation compared to PA-Cas9, a bimolecular photoswitch system, with very low leakage activity (activity produced even in the dark). Furthermore, the scMagnet system not only induced the activity of the split Cas9 endonuclease but also effectively induced the activity of the split Cre recombinase, the split Flp recombinase, and the split Vav2 (guanine nucleotide exchange factor) in a light-dependent manner (inducing more than 100 times the high dynamic range of light-dependent activation), with almost no leakage activity.

[0019] Therefore, the scMagnet system of this invention is a highly versatile system for controlling the rebinding and dissociation of various segmented proteins in an optogenetic manner.

[0020] In addition, it was confirmed that the proteins that make up scMagnet (such as pMag and nMag) can be linked by peptide linkers, which can be proteins, and the amino acid residues at the linker ends of each protein can be missing one or more.

[0021] As described above, the scMagnet system developed by the inventors is an innovative system that can function very efficiently even in environments where existing Magnet systems cannot function well, and can be used for various purposes by combining it with proteins other than Magnet, such as fluorescent proteins.

[0022] This invention is based on the above insights.

[0023] That is, the present invention relates to the following (1) to (9).

[0024] (1) A protein is formed by directly or indirectly linking the C-terminus of (a) a protein with the N-terminus of (b) a protein.

[0025] (a) A protein comprising an amino acid sequence having more than 80% sequence homology with the amino acid sequence represented by sequence number 1 or sequence number 36 by deleting X consecutive amino acid residues from the C-terminal to the N-terminal side, wherein X is an integer from 0 to 6.

[0026] (b) A protein comprising an amino acid sequence having more than 80% sequence homology with the amino acid sequence represented by sequence number 1 or sequence number 36 by deleting Y consecutive amino acid residues from the N-terminal residue to the C-terminal side, wherein Y is an integer from 0 to 55.

[0027] Wherein, when X of protein (a) is 0, Y of protein (b) is an integer from 1 to 55, and when Y of protein (b) is 0, X of protein (a) is an integer from 1 to 6.

[0028] (2) According to the protein described in (1) above, wherein, in the amino acid sequence of the protein described in (a) above, when the amino acids corresponding to the 52nd and 55th positions of the amino acid sequence represented by sequence number 1 are replaced by arginine, or the amino acids corresponding to the 16th and 19th positions of the amino acid sequence represented by sequence number 36 are replaced by arginine, in the amino acid sequence of the protein described in (b) above, the amino acid corresponding to the 52nd position of the amino acid sequence represented by sequence number 1 is replaced by aspartic acid and the amino acid corresponding to the 55th position is replaced by glycine, or the amino acid corresponding to the 16th position of the amino acid sequence represented by sequence number 36 is replaced by aspartic acid and the amino acid corresponding to the 19th position is replaced by glycine.

[0029] (3) The protein according to (1) above, characterized in that the C-terminus of the protein (a) above and the N-terminus of the protein (b) above are connected by a polypeptide.

[0030] (4) The protein according to (3) above, wherein the polypeptide is a fluorescent protein or a bioluminescent protein.

[0031] (5) The protein according to (4) above, wherein the fluorescent protein is any one of BFP, TagBFP, mTagBFP2, mBlueberry2, mCerulean, mTurquoise, ECFP, TagCFP, Rosmarius, meleCFP, mTFP1, KCY, meffCFP, GFP, YFP, Venus, mCherry and iRFP.

[0032] (6) The protein according to (4) above, wherein the bioluminescent protein is any one of NanoLu, Gaussia luciferase, Renilla luciferase and firefly luciferase.

[0033] (7) A nucleic acid that encodes the protein described in any one of (1) to (6) above.

[0034] (8) A method for controlling the distance between two proteins, comprising: a step of binding (a) protein to one of the two proteins in any one of (1) to (6) above, a step of binding (b) protein to the other of the two proteins, and a step of irradiating the protein described in any one of (1) to (6) above with light.

[0035] (9) According to the method described in (8) above, wherein the two proteins are interacting proteins.

[0036] It should be noted that the symbol “~” in this specification indicates the range of values ​​to its left and right.

[0037] By using the light-switching protein of the present invention, the binding and dissociation of proteins can be controlled very effectively in environments where existing light-switching proteins cannot function. Furthermore, the light-switching protein of the present invention can be used for various purposes, in addition to protein binding and dissociation, to add functionality to other proteins (e.g., fluorescent proteins). Attached Figure Description

[0038] Figure 1 The mechanism of scMagnet is explained. (a) The three-dimensional structure of scMagnet in the dark is shown. The N-terminus of pMag and the C-terminus of nMag are at least 6 nm apart. (b) The three-dimensional structure of scMagnet under illumination is shown. The N-terminus of pMag and the C-terminus of nMag are within 1 nm apart. (c) A schematic representation of the switching mechanism of scMagnet, which changes from an open structure to a closed structure when illuminated with blue light.

[0039] Figure 2 The results show a comparison of the activities of scMagnet (a single-molecule system) and Magnet (a two-molecule system). (a) Outline of the mechanism of action of PA-Cas9 (two-molecule system). In the dark, PA-Cas9 is split into two fragments with no catalytic activity against the target genomic region. Upon irradiation with blue light, pMag and nMag dimerize in a light-dependent manner, inducing the binding of the N-terminal and C-terminal fragments of Cas9. As a result, the structure of PA-Cas9 is completed, its activity is restored, and double-strand breaks are introduced into genomic DNA. (b) Shows the reaction of HEK293T cells catalyzed by PA-Cas9 to human... VEGFA Frequency (%) of light-induced InDel mutations at the locus. 24 hours after cell seeding, PA-Cas9 and gRNA were transfected and cultured under darkness or blue light (470±20 nm, 1.0 Wm). -2After 24 hours of maintenance, genomic DNA was recovered from the cells. The InDel mutation rate of the recovered DNA samples was determined by TIDE assay. Data are presented as mean ± sd (n=3, based on 3 independent experiments). Points represent independent data points. Full-length Cas9 and pcDNA3.1-V5-HisA (empty vector) were used as positive or negative controls, respectively. In the graphs for full-length Cas9, empty vector, and PA-Cas9, the left column represents values ​​under dark conditions, and the right column represents values ​​under light irradiation. (c) shows a summary of the mechanism of action of scPA-Cas9 (single-molecule system). Under dark conditions, scPA-Cas9 exists as a polypeptide. The N-terminal and C-terminal fragments of Cas9 are far apart, so there is no catalytic activity for the target genomic region. Upon irradiation with blue light, the single-stranded basic magnet system (scMagnet) undergoes a significant photodependent structural change, inducing the binding of the N-terminal and C-terminal fragments of Cas9. As a result, the structure of PA-Ca9 was completed, its activity was restored, and double-strand breaks were introduced into genomic DNA. (d) indicates the introduction of human DNA into HeLa cells catalyzed by scPA-Cas9 or PA-Cas9. VEGFA Frequency (%) of light-induced InDel mutations at the locus. 24 hours after cell seeding, cells were transfected with scPA-Cas9 or PA-Cas9 and gRNA, and cultured under darkness or blue light (470±20 nm, 1.0 Wm). -2 After 48 hours of maintenance, genomic DNA was recovered from the cells. The InDel mutation rate of the recovered DNA samples was determined by TIDE assay. Data are presented as mean ± sd (n=3, based on 3 independent experiments). Points represent independent data points. Full-length Cas9 and pcDNA3.1-V5-HisA (empty vector) were used as positive or negative controls, respectively. In the graphs for full-length Cas9, empty vector, PA-Cas9, and scPA-Cas9, the left bar represents the value under dark conditions, and the right bar represents the value under light conditions.

[0040] Figure 3The structure and constructs for scPA-Cas9 are described. (a) Schematic diagram of scPA-Cas9 based on the crystal structure of Cas9, gRNA, and target DNA (PDB: 4UN3), and the crystal structure of Vivid, a precursor protein of the magnetosystems (pMag and nMag), as a dimer under light irradiation (PDB: 3RH8). (b) Schematic diagram of the PA-Cas9 expression construct. CMV (cytomegalovirus promoter) represents the cytomegalovirus promoter, and NLS (nuclear localization signal) represents the nuclear localization signal. (c) Schematic diagram of the scPA-Cas9 expression construct.

[0041] Figure 4 This indicates the expression construct of the gRNA expression vector and its base sequence. (Human) VEGFA ,people DNMT1 ,people EMX1 and mice K-ras The target gRNA was prepared using the BstI site of the pU6-gRNA expression vector (Addgene: 47108) via annealed oligonucleotide cloning.

[0042] Figure 5 This refers to individuals who have had PA-Cas9 introduced into HEK293T cells. DNMT1 and people EMX1 locus The InDel mutation rate. 24 hours after cell seeding, PA-Cas9 and gRNA were transfected and cultured under darkness or blue light (470±20 nm, 1.0 Wm). -2 The assay was maintained for 24 hours. The InDel mutation rate of genomic DNA recovered from cells was determined by TIDE assay. Data are presented as mean ± sd (n=3, based on 3 independent experiments). Points represent independent data points. Full-length Cas9 and pcDNA3.1-V5-HisA (empty vector) were used as positive or negative controls, respectively. In the graphs for PA-Cas9, empty vector, and full-length Cas9, the left bar represents the value under dark conditions, and the right bar represents the value under light conditions.

[0043] Figure 6 This refers to individuals who have had scPA-Cas9 and PA-Cas9 introduced into HeLa cells. DNMT1 and people EMX1 gene seat The InDel mutation rate. 24 hours after cell seeding, cells were transfected with scPA-Cas9 or PA-Cas9 and gRNA, and cultured in the dark or under blue light (470±20 nm, 1.0 Wm). -2The culture was maintained for 48 hours. The InDel mutation rate of genomic DNA recovered from cells was determined by TIDE assay. Data are presented as mean ± sd (n=3, based on 3 independent experiments). Points represent independent data points. Full-length Cas9 and pcDNA3.1-V5-HisA (empty vector) were used as positive or negative controls, respectively. In the graphs for scPA-Cas9, PA-Cas9, empty vector, and full-length Cas9, the left bar represents values ​​under dark conditions, and the right bar represents values ​​under light conditions.

[0044] Figure 7 This demonstrates the versatility of the scMagnet system. (a) This represents a summary of the mechanism of action of scPA-Cre. scPA-Cre activated by blue light irradiation undergoes two processes... loxP Recombination of DNA sequences sandwiched at sites. CMV (cytomegalovirus promoter) represents the cytomegalovirus promoter, stop represents the polyadenylation transcription "termination" signal sequence, and Fluc represents firefly luciferase. (b) Comparison of scPA-Cre (single-molecule system) and PA-Cre (two-molecule system) based on luciferase detection. Under dark conditions or blue light irradiation (470±20nm, 1.0Wm) -2 ), will transiently transfect with scPA-Cre or PA-Cre and loxP -stop- loxP Luciferase assays in COS-7 cells, using Fluc reporter gene expression as an indicator, were performed 24 hours later. The ratio of scPA-Cre encoded DNA or PACre encoded DNA to total transfection was 4.8%. Data are mean ± sd (n=4, based on 3 independent experiments). Points are independent data points. In the graphs for PA-Cre and scPA-Cre, the left bar represents values ​​under darkness, and the right bar represents values ​​under light. (c) shows a summary of the mechanism of action of scPA-Flp. scPA-Flp activated by blue light occurs through two... FRT Recombination of DNA sequences sandwiched at sites. (d) Comparison of scPA-Flp (single-molecule system) and PA-Flp (two-molecule system) based on luciferase detection. Under dark conditions or blue light irradiation (470±20nm, 1.0Wm) -2 ), will transiently transfect with scPA-Flp or PA-Flp and FRT -stop- FRTLuciferase assays were performed on COS-7 cells to measure Fluc expression as an indicator of Fluc reporter gene expression for 24 hours. The ratio of scPA-Cre encoded DNA or PA-Cre encoded DNA to total transfection was 4.8%. Data are presented as mean ± sd (n=4, based on 3 independent experiments). Points represent independent data points. In the PA-Flp and scPA-Flp graphs, the left bar represents values ​​under dark conditions, and the right bar represents values ​​under light conditions.

[0045] Figure 8 The structure and constructs used to create scPA-Cre are described. (a) Schematic diagram of scPA-Cre crystal structure based on Cre recombinase (PDB: 1CRX) and Vivid light-irradiated dimer (PDB: 3RH8). (b) Schematic diagram of the PA-Cre expression construct. CMV (cytomegalovirus promoter) represents the cytomegalovirus promoter, NLS (nuclear localization signal) represents the nuclear localization signal, and P2A represents the self-cleaving peptide. (c) Schematic diagram of the scPA-Cre expression construct.

[0046] Figure 9 The results of the activity evaluation of scPA-Cre are shown. (a) Comparison of scPA-Cre and PA-Cre based on luciferase assay. Under dark conditions or blue light irradiation (470±20 nm, 1.0 W / m²), the results are presented. -2 ), will transiently transfect with scPA-Cre or PA-Cre and loxP -stop- loxP Luciferase assays for Fluc expression in COS-7 cells, using the Fluc reporter gene as an indicator, were performed for 24 hours. The ratio of scPA-Cre encoded DNA or PA-Cre encoded DNA to total transfection was 9.1%. Data are presented as mean ± SD (n=12, based on 3 independent experiments). Points represent independent data points. Full-length Cre and pcDNA3.1 (empty vector) served as positive or negative controls, respectively. In the graphs for PA-Cre, scPA-Cre, empty vector, and full-length Cre, the left column represents values ​​under darkness, and the right column represents values ​​under light. (b) Light intensity dependence of scPA-Cre and PA-Cre. Under darkness or blue light (470±20nm, 0.01~3.3Wm) -2 ), will transiently transfect with scPA-Cre or PA-Cre and loxP -stop- loxPLuciferase assays in COS-7 cells, using Fluc reporter gene expression as an indicator, were performed over 24 hours. The ratio of scPA-Cre encoded DNA or PA-Cre encoded DNA to total transfection was 9.1%. Data are mean ± sd (n=12, based on 3 independent experiments). Points are independent data points. (c) is a graph showing the experimental timeline in relation to short-duration and continuous blue light irradiation. (d) Blue light irradiation time at different durations (100Wm) -2 30 seconds to 3 minutes; 1.0 Wm -2 A comparison of the responses of scPA-Cre or PA-Cre (30 min to 24 h). Data are presented as mean ± SD (n=12, based on 3 independent experiments). Points are independent data points.

[0047] Figure 10 The structure and constructs used to prepare scPA-Flp are described. (a) Schematic diagram of the crystal structure of scPA-Flp based on Flp recombinase (PDB: 1FLO) and the Vivid light-irradiated dimer (PDB: 3RH8). (b) Schematic diagram of the expression construct of PA-Flp. pMagHigh1 is the magnet of the magnet system, which has high affinity and long photoperiod. CMV (cytomegalovirus promoter) represents the cytomegalovirus promoter, and NLS (nuclear localization signal) represents the nuclear localization signal. (c) Schematic diagram of the expression construct of scPA-Flp.

[0048] Figure 11 The results of the scPA-Flp activity evaluation are shown. (a) Comparison of scPA-Flp and PA-Flp based on luciferase assay. Under dark conditions or blue light irradiation (470±20 nm, 1.0 W / m²), the results are presented. -2 ), will transiently transfect with scPA-Flp or PA-Flp and FRT -stop- FRTLuciferase assays for Fluc expression in COS-7 cells, using Fluc reporter gene as an indicator, were performed for 24 hours. The ratio of scPA-Flp-encoded DNA or PA-Flp-encoded DNA to total transfection was 9.1%. Data are presented as mean ± SD (n=4, based on 3 independent experiments). Points are independent data points. Full-length Flp and pcDNA3.1 (empty vector) were used as positive or negative controls, respectively. In the graphs for PA-Flp, scPA-Flp, empty vector, and full-length Flp, the left column represents values ​​under darkness, and the right column represents values ​​under light. (b) Light intensity dependence of scPA-Flp and PA-Flp. Under darkness or blue light (470±20 nm, 0.01–3.3 Wm) -2 ), will transiently transfect with scPA-Flp or PA-Flp and FRT -stop- FRT The luciferase assay in COS-7 cells, using Fluc reporter gene expression as an indicator, was performed over 24 hours. Data are presented as mean ± sd (n=4, based on 3 independent experiments). Points are independent data points. (c) is a graph showing the experimental timeline in relation to short-duration and continuous blue light exposure. (d) Blue light exposure time at different durations (100 Wm) -2 30 seconds to 3 minutes; 1.0 Wm -2 The responses of scPA-Flp or PA-Flp were compared (30 min to 24 h). Data are presented as mean ± SD (n=4, based on 3 independent experiments). Points are independent data points. In the graphs for PA-Flp, scPA-Flp, and full-length Flp, the left bar represents the value under darkness, and the right bar represents the value under illumination.

[0049] Figure 12 This indicates the results of evaluating the activities of scPA-Cre and scPA-Flp in NIH3T3 cells. (a) Comparison of scPA-Cre and PA-Cre activities based on luciferase assay. Under dark conditions or blue light irradiation (470±20nm, 1.0Wm) -2 ), will transiently transfect with scPA-Cre or PA-Cre and loxP -stop- loxPLuciferase assays of Fluc reporter gene expression in NIH3T3 cells were performed for 24 hours. The ratio of scPA-Cre encoded DNA or PA-Cre encoded DNA to total transfection was 4.8%. Data are mean ± sd (n=4, based on 3 independent experiments). Points are independent data points. In the graphs for PA-Cre and scPA-Cre, the left bar represents the value under darkness, and the right bar represents the value under light. (b) Comparison of scPA-Flp and PA-Flp based on luciferase assay. Under darkness or blue light (470±20nm, 1.0Wm) -2 ), will transiently transfect with scPA-Flp or PA-Flp and FRT -stop- FRT Luciferase assays were performed on NIH3T3 cells to measure Fluc expression as an indicator of the Fluc reporter gene, over a period of 24 hours. The ratio of scPA-Cre encoded DNA or PA-Cre encoded DNA to total transfection was 4.8%. Data are presented as mean ± SD (n=4, based on 3 independent experiments). Points represent independent data points. In the PA-Flp and scPA-Flp graphs, the left bar represents values ​​under dark conditions, and the right bar represents values ​​under light conditions.

[0050] Figure 13 The results show the effects of linker and amino acid deletions in pMag on the activity of scMagnet. (a) shows the results of comparing the activity of directly linked scPA-Cre with that of scPA-Cre linked by linkers of various lengths using luciferase assay. (b) shows the results of comparing the activity of directly linked scPA-Cre with that of scPA-Cre mutants with 1–6 amino acid deletions on the C-terminal side of pMag and linked by linkers of various lengths using luciferase assay. Results were obtained under darkness or blue light irradiation (470±20 nm, 1.0 Wm). -2 ), transiently transfected with scPA-Cre or scPA-Cre mutants and loxP -stop- loxP Luciferase assays in COS-7 cells, using Fluc reporter gene expression as an indicator, were performed over 24 hours. Data are presented as mean ± sd (n=4, based on 3 independent experiments). Points represent independent data points. In the graphs for each scPA-Cre or scPA-Cre mutant, the upper bars represent values ​​under dark conditions, and the lower bars represent values ​​under light conditions.

[0051] Figure 14This represents the results of a study on the suitability of the adapter for scMagnet using scPA-Cas9. (a) Insertion of a flexible adapter (GSGGGSGGGSGGGSGGGSGS; SEQ ID NO. 34) or a rigid adapter (GSAEAAAKAGSAEAAAKAGS; SEQ ID NO. 35) between pMag and nMagHigh1. "No adapter" indicates that pMag and nMagHigh1 are directly connected. CMV (cytomegalovirus promoter) represents the cytomegalovirus promoter, and NLS (nuclear localization signal) represents the nuclear localization signal. (b) 5-11 cells (from LSL- KrasG12D / + LSL-Trp53 R172H / + ; ptf1a- Cre (KPC) mouse pancreatic cancer cell line in mice K-ras Determination of light-induced InDel mutations at loci caused by scPA-Cas9 and PA-Cas9. Twenty-four hours after cell seeding, cells were transfected with scPA-Cas9 or PA-Cas9 and gRNA, and cultured under darkness or blue light (470±20 nm, 1.0 Wm). -2 The culture was maintained for 48 hours. The InDel mutation rate of genomic DNA recovered from cells was determined by TIDE assay. Data are presented as mean ± sd (n=3, based on 3 independent experiments). Points represent independent data points. Full-length Cas9 and pcDNA3.1-V5-HisA (empty vector) were used as positive or negative controls, respectively. In the graphs for scPA-Cas9 (flexible, rigid, and adapter-free), PA-Cas9, empty vector, and full-length Cas9, the left column represents values ​​under dark conditions, and the right column represents values ​​under light conditions.

[0052] Figure 15 The results represent the effects of amino acid deletions in nMag within scMagnet on its activity. The results also represent the comparison of the activity of directly linked scPA-Cre with that of scPA-Cre mutants linked to pMag, obtained by luciferase assay, with 1–19 amino acids deleted from the N-terminus of nMag. Results were obtained under darkness or blue light irradiation (470±20 nm, 1.0 Wm). -2 ), transiently transfected with scPA-Cre or scPA-Cre mutants and loxP -stop- loxPLuciferase assays in COS-7 cells, using Fluc reporter gene expression as an indicator, were performed over 24 hours. Data are presented as mean ± sd (n=4, based on 3 independent experiments). Points represent independent data points. In the graphs for each scPA-Cre or scPA-Cre mutant, the upper bars represent values ​​under dark conditions, and the lower bars represent values ​​under light conditions.

[0053] Figure 16 This indicates the results of investigating the Cre recombination activity of the scPA-Cre mutant of nMag with a staged deletion of amino acids at the N-terminus. scPA-Cre mutants were prepared by introducing the nMag mutant containing only the Latch-NCap-Hinge, and scPA-Cre mutants of the nMag mutant with deletions up to amino acid residue 119 (Δ119), amino acid residue 71 (Δ71), amino acid residue 56 (Δ56), or amino acid residue 35 (Δ35) at the N-terminus. Cre recombination activity was measured. Figure a shows a schematic diagram of the structure of each nMag mutant, and figure b shows the results of the Cre recombination activity determination. Results were obtained under darkness or blue light irradiation (470±20 nm, 1.0 Wm). -2 Under these conditions, transiently transfected individuals with scPA-Cre (pMag+nMag) or scPA-Cre mutants and loxP -stop- loxP The luciferase assay in COS-7 cells, using Fluc reporter gene expression as an indicator, was performed over 24 hours. Data are presented as mean ± sd (n=4, based on 3 independent experiments). Points represent independent data points. In the graph in b, the upper bars represent values ​​under dark conditions, and the lower bars represent values ​​under light conditions.

[0054] Figure 17This report presents the results of investigating the Cre recombination activity of scPA-Cre mutants created by linking mutants with various combinations of deletions at the C-terminus of pMag and the N-terminus of nMag. pMag mutants with deletions of 1 (pMag-CtermΔ1), 2 (pMag-CtermΔ2), 5 (pMag-CtermΔ5), or 6 (pMag-CtermΔ6) amino acids at the C-terminus of pMag, and nMag mutants with deletions of 1 (nMag-NtermΔ1), 2 (nMag-NtermΔ2), 9 (nMag-NtermΔ9), or 19 (nMag-NtermΔ19) amino acids at the N-terminus of nMag, were created and linked together to form various scPA-Cre mutants. Their Cre recombination activity was then measured. Results were obtained under darkness or blue light irradiation (470±20 nm, 1.0 Wm). -2 Transiently transfected with scPA-Cre (pMag+nMag) or scPA-Cre mutants and loxP -stop- loxP The luciferase assay for Fluc expression in COS-7 cells, using the Fluc reporter gene as an indicator, was performed over 24 hours. Data are presented as mean ± sd (n=4, based on 3 independent experiments). Points represent independent data points. In the graphs, the upper bars represent values ​​under dark conditions, and the lower bars represent values ​​under light conditions.

[0055] Figure 18 The results represent the evaluation of the light-dependent recombination cascade of the full-length Cre and scPA-Flp combination. (a) shows a summary of the recombination cascade using multiple gene expression stages. (b) COS-7 cells transfected with cDNA as shown in (c) are subjected to light in the dark or under blue light irradiation (470±20nm, 1.0Wm). -2 The culture was maintained for 24 hours, followed by luciferase detection. Data are presented as mean ± SD (n=4, based on 3 independent experiments). Points represent independent data points. Full-length Cas9 and pcDNA3.1-V5-HisA (empty vector) were used as positive or negative controls, respectively. In the graphs for empty vector and full-length Cre, the left column represents values ​​under darkness, and the right column represents values ​​under light. (c) shows the transfection conditions for the luciferase detection illustrated in (b).

[0056] Figure 19This section presents the results of in vivo evaluation of the light-dependent recombination cascade of the full-length Cre and scPA-Flp combination. (a) shows a summary of the recombination cascade based on multiple gene expression stages. (b) shows the timeline of the in vivo experiments. (c) shows the results obtained from CreER... T2 The intracellular progression of the initial scPA-Flp recombination cascade. Rosa26 -CreER T2 After gene knock-in mice were injected with tamoxifen or corn oil, the encoded gene was transiently injected into the mice via HTV (hydrodynamic tail vein) injection. loxP -stop- loxP cDNA of scPA-Flp and FRT -stop- FRT mKate2 reporter gene, in the dark or under blue light (470±20nm, 132Wm) -2 Mice were fed for 18 hours. Liver samples were removed from mice after 18 hours and subjected to fluorescence imaging. Images represent typical results obtained from three measurements. Scale bar: 1 mm.

[0057] Figure 20 This indicates the results of evaluating scPA-Flp activity in vivo. (a) shows the experimental timeline. Rosa26 -CreER T2 Gene knock-in mice were injected intraperitoneally with 200 μL of tamoxifen (27 mM) or corn oil. Six hours later, the mice were injected with the gene knock-in gene via the HTV (hydrodynamic tail vein) injection method. loxP -stop- loxP cDNA of scPA-Flp and FRT -stop- FRT The mKate2 reporter gene, 6 hours after cDNA injection, was tested under darkness or blue light (470±20nm, 132Wm). -2 (a) Mice were fed for 18 hours. Liver samples were removed from mice after 18 hours and subjected to fluorescence imaging. (b) indicates... Figure 16 Image c is a different image from the previous one. The scale bar is 1 mm.

[0058] Figure 21Schematic diagrams illustrating the structure and mechanism of action of scPA-Vav. (a) Schematic diagram of the crystal structure of scPA-Vav based on the light-irradiated dimer (PDB: 3RH8) of mouse Vav2 GEF9 and Vivid. (b) Schematic diagram illustrating the mechanism of action of scPA-Vav during cell stretching. Vav2 is a Rho family guanine exchanger that converts GDP to GTP, activating Rac1 GTPase in various cells. GAP represents GTPase-activated protein, and PAK represents p21-associated kinase.

[0059] Figure 22 The results show the findings from studies investigating the reversibility of scMagnet's function. (a) Schematic diagram of scPA-Vav under blue light (BL) irradiation. (b) Schematic diagram of the scPA-Vav construct. Lifeact-mCherry was used as a biosensor to detect actin polymerization in the stretching assay of cellular regions. P2A represents the self-cleaving peptide. (c) Results show the detection of light-dependent stretching activity of cellular regions induced by scPA-Vav. Fluorescence images of NIH 3T3 cells expressing Lifeact-mCherry and scPA-Vav2 were acquired 15 minutes before, during, and 15 minutes after blue light irradiation. Blue light irradiation was performed using a 488 nm, 1 mW laser. Binary images of the cells are shown in the right panel. Typical results are shown based on four independent experiments. (d) Results show the quantification of the stretching regions of cells expressing scPA-Vav2. Independent data values ​​were plotted (n=4, based on four independent experiments). (e) Quantitative results of the stretched regions of scPA-Vav2 expressing cells in the presence of EHop-016 (a Rac GTPase inhibitor, 1 μM). Independent data values ​​were plotted (n=4, based on 4 independent experiments). (f) is a graph illustrating the inhibition of Rac1 binding to Rho GEF Vav2 by EHop-016. (g) is a Kymograph of the cellular regions along the line shown in the left panel. Results represent typical experimental results selected from 4 independent experiments. ***P < 0.001 based on Welch's two-tailed t-test. ns indicates no significant difference. Scale bar is 10 μm.

[0060] Figure 23This represents the results of quantifying cell stretching induced by scPA-Vav and 2PscPA-Vav. Fluorescence images of NIH 3T3 cells expressing Lifeact-mCherry, and scPA-Vav or 2PscPA-Vav, were obtained 15 minutes before (dark), during (before), and 15 minutes after (after) blue light irradiation. Blue light irradiation was performed using a 488 nm, 1 mW laser in single-photon excitation experiments and an 808 nm, 25 mW laser in two-photon excitation experiments. Binary images of the cells are shown in the right panel. Typical results are shown based on four independent experiments. Fluorescence images were converted to binary data using ImageJ software. Using the binary data, the ratio of "dark" cell regions to "before" cell regions and the ratio of "before" cell regions to "after" cell regions were calculated.

[0061] Figure 24 This represents the resolution of light-dependent cellular stretching induced by scPA-Vav. Fluorescence images of NIH 3T3 cells expressing Lifeact-mCherry and scPA-Vav were acquired 15 minutes before, during, and 15 minutes after blue light irradiation. Blue light irradiation was performed using a 488 nm, 1 mW laser. Fluorescence images were converted to binary data using ImageJ software. Using the binary data, the ratio of the pre-irradiation cellular region to the dark cellular region (dark) and the ratio of the post-irradiation cellular region to the pre-irradiation cellular region (BL) were calculated. The calculated cell stretching rates were used to fabricate... Figure 22 Charts for d and e. All scale bars are 10 μm.

[0062] Figure 25The results show the findings of an investigation into the operability of the scMagnet system based on two-photon excitation. (a) Schematic diagram of the mechanism of action of 2PscMagnet. mTagBFP2 is inserted between pMag and nMag (2PscMagnet). 2PscMagnet can be activated by two-photon excitation via fluorescence resonance energy transfer (FRET) from mTagBFP2 to flavinadenine dinucleotide (FAD) bound to pMag and nMag. (b) Three-dimensional structure of 2PscMagnet. The distances between the chromophore of mTagBFP2 (FRET donor) and the FAD (FRET acceptor) of pMag and nMag are approximately 3 nm and 4 nm, respectively. This distance is sufficient for FRET to occur. (c) Schematic diagram of the structure of 2PscPA-Vav. Based on crystal structure information, the N-terminal amino acid residues (aa 1-6) and C-terminal amino acid residues (aa 229-237) of mTagBPF2 were shortened to the maximum extent and inserted into scMagnet. (d) shows the results of quantifying the stretched regions of 2PscPA-Vav-expressing cells stretched by single-photon excitation. The stretching of light-dependent cell regions was induced by irradiation with a 488 nm, 1 mW laser. For the data, independent data values ​​were plotted (n=4, based on 4 independent experiments). (e) The stretching of light-dependent cell regions was induced by two-photon (2P) excitation of 2PsPA-Vav. Fluorescence images of NIH 3T3 cells expressing Lifeact-mCherry and 2PscPA-Vav2 were obtained 15 min before, during, and 15 min after two-photon excitation. Two-photon excitation was performed using an 808 nm, 25 mW laser. The binary images of the cells are shown in the right panel. Typical results are shown based on 4 independent experiments. (f and g) represent the results of quantifying the stretched regions of cells expressing 2PscPA-Vav2 (f) or scPA-Vav2 (g). Light-dependent stretching of cellular regions was induced using 2PsPA-Vav or scPA-Vav2 with an 808 nm, 25 mW laser. For the data, independent data values ​​were plotted (n=4, based on 4 independent experiments). ***P < 0.001 was based on Welch's two-tailed t-test. ns indicates no significant difference. Scale bar is 10 μm.

[0063] Figure 26This represents the results obtained from the analysis of light-dependent stretching of cellular regions induced by 2PscPA-Vav. Fluorescence images of NIH 3T3 cells expressing Lifeact-mCherry and 2P scPA-Vav were obtained 15 minutes before (dark), during (before), and 15 minutes after (after) light exposure. Light exposure was performed with a 488 nm, 1 mW laser for single-photon excitation (a) and with an 808 nm, 25 mW laser for two-photon excitation (b). Fluorescence images were converted to binary data using ImageJ software. Using the binary data, the ratio of the "dark" cellular region to the "before" cellular region and the ratio of the "before" cellular region to the "after" cellular region were calculated. The calculated cell stretching rates were used to fabricate... Figure 25 Charts for d and f. All scale bars are 10 μm.

[0064] Figure 27 This represents the results obtained from the analysis of light-dependent stretching of cellular regions induced by scPA-Vav. Fluorescence images of NIH 3T3 cells expressing Lifeact-mCherry and scPA-Vav were acquired 15 minutes before (dark), during (before) and 15 minutes after (after) light irradiation. Irradiation was performed using an 808 nm, 25 mW laser. Fluorescence images were converted to binary data using ImageJ software. Using the binary data, the ratio of "dark" cell regions to "before" cell regions and the ratio of "before" cell regions to "after" cell regions were calculated. The calculated cell stretching rates were used to fabricate... Figure 25 A chart for g. All scale bars are 10 μm.

[0065] Figure 28 The nucleotide sequence representing pcDNA3.1-V5-HisA-PA-Cas9_N-term (Sequence No. 18).

[0066] Figure 29 The nucleotide sequence representing pcDNA3.1-V5-HisA-PA-Cas9_C-term (sequence number 19).

[0067] Figure 30 The nucleotide sequence representing pcDNA3.1-V5-HisA-scPA-Cas9 (flexible linker) (Sequence number 20).

[0068] Figure 31The nucleotide sequence representing pcDNA3.1-V5-HisA-scPA-Cas9 (rigid linker) is 21.

[0069] Figure 32 The nucleotide sequence representing pcDNA3.1-V5-HisA-scPA-Cas9 (direct binding) is sequence number 22.

[0070] Figure 33 The nucleotide sequence representing pcDNA3.1-PA-Cre (sequence number 23).

[0071] Figure 34 The nucleotide sequence representing pcDNA3.1-scPA-Cre (sequence number 24).

[0072] Figure 35 The nucleotide sequence representing pcDNA3.1-loxP-stop-loxP-Fluc (Sequence number 25).

[0073] Figure 36 The nucleotide sequence representing pcDNA3.1-PA-Flp_N-term (Sequence number 26).

[0074] Figure 37 The nucleotide sequence representing pcDNA3.1-PA-Flp_C-term (Sequence number 27).

[0075] Figure 38 The nucleotide sequence representing pcDNA3.1-scPA-Flp (sequence number 28).

[0076] Figure 39 The nucleotide sequence representing pcDNA3.1-FRT-stop-FRT-Fluc (Sequence number 29).

[0077] Figure 40 The nucleotide sequence (sequence number 30) represents pcDNA3.1-FRT-stop-FRT-Akaluc-mKate2.

[0078] Figure 41 The nucleotide sequence representing pcDNA3.1-loxP-stop-loxP-scPA-Flp is (sequence number 31).

[0079] Figure 42 The nucleotide sequence representing pEF6-Lifeact-mCherry-P2A-scPA-Vav (Sequence number 32).

[0080] Figure 43The nucleotide sequence representing pEF6-Lifeact-mCherry-P2A-2PscPA-Vav is (sequence number 33). Detailed Implementation

[0081] The following describes the methods for implementing the present invention. It should be noted that, unless otherwise specified, "this embodiment" refers to all embodiments described in this specification.

[0082] The first embodiment is a light-switching protein, which, unlike existing light-switching proteins composed of two separate proteins, is a light-switching protein composed of a single polypeptide. The "light-switching protein" involved in this embodiment is a protein formed by directly linking two proteins selected from VVD or VVD mutants (hereinafter, unless otherwise specified, "VVD protein" refers to VVD or VVD mutants), or indirectly linking them through a linker of a single amino acid residue or peptide.

[0083] Depending on the situation, the VVD protein may have one or more consecutive amino acid residues deleted from the terminal amino acid residues on the side linked to other VVD proteins toward the opposite terminal side. For example, when the light-switching protein according to this embodiment is a wild-type VVD and a VVD mutant (denoted as VVD1) linked by a polypeptide linker L (Nter-VVD-L-VVD1-Cter), one or more consecutive amino acid residues may be deleted from the C-terminal residues of the wild-type VVD toward the N-terminal side, and / or one or more consecutive amino acid residues may be deleted from the N-terminal residues of VVD1 toward the C-terminal side. Here, in the deletion of "one or more" amino acid residues, "a plurality of" is, for example, 2 to 30, preferably 2 to 20, and more preferably 2 to 10. In the light-switching protein according to this embodiment, by deleting the amino acid residues in the terminal region of the VVD protein bound to the linker as described above, the leakage activity of the light-switching protein in the dark can be reduced.

[0084] When two VVD proteins constituting the photo-switching protein of this embodiment are indirectly linked, the two VVD proteins may be linked by a linker consisting of a single amino acid residue or a peptide. When the linker is a peptide, it may be an oligopeptide (e.g., with about 10 or fewer amino acids), a polypeptide (e.g., with about 10 to 100, 10 to 50, or 10 to 30 amino acids), or a protein. When the linker is an oligopeptide or polypeptide and is not a peptide with a specific structure, the amino acid constituting the linker is not particularly limited; examples include glycine (G), serine (S), alanine (A), and threonine (T). In addition, in the case of proteins, there are no particular limitations. For example, it can be fluorescent proteins (e.g., BFP, TagBFP, mTagBFP2, mBlueberry2, mCerulean, mTurquoise, ECFP, TagCFP, Rosmarius, meleCFP, mTFP1, KCY, meffCFP, GFP, YFP, Venus, mCherry, and iRFP, etc.) and bioluminescent proteins (e.g., NanoLuc, Gaussian luciferase, Renilla luciferase, and firefly luciferase, etc.).

[0085] The photoswitch protein of this embodiment exhibits activity where its structure undergoes a significant change upon light irradiation, with VVD proteins on both sides of the connector binding to each other, and dissociating in the dark (without light irradiation). When two interacting proteins are pre-linked to the two VVD proteins constituting the photoswitch protein of this embodiment (i.e., the N-terminal and C-terminal sides of the photoswitch protein), the photoswitch protein can be brought close together by receiving light irradiation, enabling them to interact. When light irradiation is stopped, the two fragments can dissociate. Here, "two interacting proteins" can refer to two protein fragments that were originally a single protein but have been split. Therefore, when fragments of a protein are bound to the N-terminal and C-terminal sides of the photoswitch protein of this embodiment, the rebinding and dissociation of these fragments can be controlled by switching light irradiation on / off, allowing for control of the activity of the protein (before splitting).

[0086] As described above, the VVD protein in this embodiment includes not only wild-type VVD (Sequence No. 1), but also its mutants. Besides the VVD mutants described later, it can also be a VVD mutant (Sequence No. 36) with amino acid residues from position 1 to 36 missing from the amino acid sequence represented by Sequence No. 1. It has been reported that when VVD is expressed, the expression level increases if 36 amino acid residues are deleted from its N-terminus (Zoltowski et al., Biochemistry 47: 7012-7019 2008). Therefore, as the VVD protein in this embodiment, a protein with the amino acid mutation described later can be appropriately introduced into the VVD protein composed of the amino acid sequence represented by Sequence No. 1 or Sequence No. 36.

[0087] More specifically, the light-switching protein involved in this embodiment is a protein formed by directly or indirectly connecting the C-terminus of a protein to the N-terminus of a protein (a) and (b) the N-terminus of a protein.

[0088] (a) A protein is an amino acid sequence having more than 80% sequence homology with the amino acid sequence represented by sequence number 1 or sequence number 36, wherein the amino acid sequence is missing X consecutive amino acid residues from the C-terminal residue to the N-terminal side, and X is an integer from 0 to 6.

[0089] (b) A protein is an amino acid sequence that has more than 80% sequence homology with the amino acid sequence represented by sequence number 1 or sequence number 36, wherein the amino acid sequence is missing Y consecutive amino acid residues from the N-terminal residue to the C-terminal side, and Y is an integer from 0 to 30.

[0090] Here, the protein composed of the amino acid sequence represented by sequence number 1 shown below is Vivid (VVD) from Neurospora Crassa, and the protein composed of the amino acid sequence represented by sequence number 36 is a VVD mutant composed of the amino acid sequence obtained by deleting amino acids from position 1 to position 36 in the amino acid sequence represented by sequence number 1.

[0091] Serial Number 1:

[0092] MSHTVNSSTMNPWEVEAYQQYHYDPRTAPTANPLFFHTLYAPGGYDIMGYLIQIMNRPNPQVELGPVDTSCALILCDLKQKDTPIVYASEAFLYMTGYSNAEVLGRNCRFLQSPDGMVKPKSTRKYVDSNTINTMRKAIDRNAEVQVEVVNFKKNGQRFVNFLTMIPVRDETGEYRYSMGFQCETE

[0093] Serial number 36:

[0094] HTLYAPGGYDIMGYLIQIMNRPNPQVELGPVDTSCALILCDLKQKDTPIVYASEAFLYMTGYSNAEVLGRNCRFLQSPDGMVKPKSTRKYVDSNTINTMRKAIDRNAEVQVEVVNFKKNGQRFVNFLTMIPVRDETGEYRYSMGFQCETE

[0095] In the phrase "an amino acid sequence in which X consecutive amino acid residues are missing from the C-terminal to the N-terminus of the amino acid sequence represented by Serial No. 1 or Serial No. 36", for example, when X is 0, the sequence is one in which no amino acid residues are missing in the amino acid sequence represented by Serial No. 1 or Serial No. 36. Alternatively, when X is 20, the sequence is one in which 20 consecutive amino acid residues are missing from the C-terminal to the N-terminus of the amino acid sequence represented by Serial No. 1 or Serial No. 36. More specifically, for example, "an amino acid sequence in which 20 consecutive amino acid residues are missing from the C-terminal to the N-terminus of the amino acid sequence represented by Serial No. 1" refers to a sequence consisting of consecutive amino acid residues from position 1 to position 166 in the amino acid sequence represented by Serial No. 1. Here, X is an integer from 0 to 30, preferably an integer from 0 to 20, more preferably an integer from 0 to 15, and even more preferably an integer from 0 to 6.

[0096] In the phrase "an amino acid sequence in which Y consecutive amino acid residues are missing from the N-terminal residue to the C-terminus of the amino acid sequence represented by Serial No. 1 or Serial No. 36", for example, when Y is 0, the sequence is one in which no amino acid residues are missing in the amino acid sequence represented by Serial No. 1 or Serial No. 36. Alternatively, when Y is 20, the sequence is one in which 20 consecutive amino acid residues are missing from the N-terminal residue to the C-terminus of the amino acid sequence represented by Serial No. 1 or Serial No. 36. More specifically, for example, "an amino acid sequence in which 20 consecutive amino acid residues are missing from the N-terminal residue to the C-terminus of the amino acid sequence represented by Serial No. 1" refers to a sequence consisting of consecutive amino acid residues from position 21 to position 186 in the amino acid sequence represented by Serial No. 1. Here, Y is an integer from 0 to 70, preferably an integer from 0 to 60, more preferably an integer from 0 to 55, and even more preferably an integer from 0 to 35.

[0097] In proteins (a) and (b) constituting the light-switching protein of this embodiment, when X of protein (a) is 0, Y of protein (b) can be an integer from 1 to 30, and when Y of protein (b) is 0, X of protein (a) can be an integer from 1 to 30. That is, at least one of the linker-side terminals (i.e., the terminal of the other VVD protein or the side where the linker binds) of the light-switching protein of this embodiment may be missing 1 to 30 amino acid residues.

[0098] In addition, in this embodiment, "protein with more than 80% sequence homology" refers to a protein that has more than 80%, more than 81%, more than 82%, more than 83%, more than 84%, more than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% sequence homology with "the amino acid sequence represented by sequence number 1 or 36 which has more than 80%, more than 81%, more than 82%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% sequence homology, or a protein with 100% sequence homology. Here, "proteins with more than 80% sequence homology" refers to mutants of proteins whose amino acid sequences are represented by sequence number 1 or sequence number 36, excluding proteins with 100% sequence homology. The VVD mutant constituting the light-switching protein of this embodiment is not particularly limited as long as it has the property of binding to other VVD proteins constituting the light-switching protein of this embodiment upon light irradiation and releasing (dissociating) this binding in the dark. For example, the VVD mutant described below is preferred.

[0099] The preferred mutants mentioned above include, for example, the mutants disclosed in Patent Document 1. In this embodiment, when the photoswitching protein, i.e., the protein belonging to (a) or (b) above, is a VVD mutant, it is preferable that in one VVD mutant (denoted as "VVD mutant 1"), at least one amino acid at the binding surface with the other VVD mutant (denoted as "VVD mutant 2") is replaced by an amino acid with a positive charge on its side chain, whereas in VVD mutant 2, at least one amino acid at the binding surface with VVD mutant 1 is replaced by an amino acid with a negative charge on its side chain. Such VVD mutants exhibit high binding affinity to each other.

[0100] It should be noted that in this specification, "amino acid" is used in the broadest sense, including not only natural amino acids but also their derivatives and artificial amino acids. For example, the term "amino acid" in this specification includes not only natural amino acids but also non-natural amino acids. Furthermore, amino acids whose main chain structure differs from their natural form, amino acids whose side chain structure differs from their natural form, amino acids with extra methylene groups in their side chains, and amino acids in which the carboxylic acid functional group in the side chain is replaced by a sulfonic acid group are also included in the term "amino acid".

[0101] The amino acids with positive charges on their side chains can be either natural or non-natural. Examples of natural amino acids include lysine, arginine, and histidine. Similarly, amino acids with negative charges on their side chains can also be either natural or non-natural. Examples of natural amino acids include aspartic acid and glutamic acid.

[0102] For the VVD mutant constituting the photoswitch protein according to this embodiment, in at least one VVD mutant, at least one amino acid located in the flavin adenine dinucleotide (FAD) binding domain (FAD binding domain) of the cofactor can be replaced. The FAD binding domain can be appropriately determined by those skilled in the art through protein crystallography or the like. More than one amino acid in the FAD binding domain can be replaced. The number of amino acids replaced is not particularly limited, for example, it can be 1, 2, 3, 4, 5, 6, 7, 8, or 9. In addition, as long as the binding and dissociation between the VVDs or VVD mutants constituting the photoswitch protein proceeds at the desired rate, the amino acid in the FAD binding domain can be replaced with any amino acid.

[0103] The VVD mutant involved in this embodiment will be described in further detail.

[0104] In this specification, when determining the position of the amino acid sequence of the VVD mutant, "the amino acid corresponding to the Z-position on the amino acid sequence represented by sequence number 1 or sequence number 36" refers to the amino acid in the VVD mutant sequence that corresponds to the Z-position on the amino acid sequence of sequence number 1 or sequence number 36, when compared in a manner that maximizes the similarity between the amino acid sequence represented by sequence number 1 or sequence number 36 and the amino acid sequence of the VVD mutant.

[0105] When the photoswitching protein involved in this embodiment includes VVD mutant 1 and VVD mutant 2, for example, in VVD mutant 1, the amino acid corresponding to position 52 of the amino acid sequence represented by sequence number 1 can be an amino acid with a positive charge on its side chain, and in VVD mutant 2, the amino acid corresponding to position 52 of the amino acid sequence represented by sequence number 1 can be an amino acid with a negative charge on its side chain. Alternatively, in VVD mutant 1, the amino acid corresponding to position 16 of the amino acid sequence represented by sequence number 36 can be an amino acid with a positive charge on its side chain, and in VVD mutant 2, the amino acid corresponding to position 16 of the amino acid sequence represented by sequence number 36 can be an amino acid with a negative charge on its side chain. It should be noted that when the two VVD proteins used in this embodiment are a combination of mutants, it can be a combination of a mutant consisting of an amino acid sequence with a mutated sequence introduced into the amino acid sequence represented by sequence number 1 and a mutant consisting of an amino acid sequence with a mutated sequence introduced into the amino acid sequence represented by sequence number 36 (the same applies to combinations of mutants described below).

[0106] When the photoswitching protein involved in this embodiment includes VVD mutant 1 and VVD mutant 2, for example, in VVD mutant 1, the amino acid corresponding to position 55 of the amino acid sequence represented by sequence number 1 can be an amino acid with a positive charge on its side chain, and in VVD mutant 2, the amino acid corresponding to position 55 of the amino acid sequence represented by sequence number 1 can be an amino acid with a negative charge on its side chain. Alternatively, in VVD mutant 1, the amino acid corresponding to position 19 of the amino acid sequence represented by sequence number 36 can be an amino acid with a positive charge on its side chain, and in VVD mutant 2, the amino acid corresponding to position 19 of the amino acid sequence represented by sequence number 36 can be an amino acid with a negative charge on its side chain.

[0107] When the light-switching protein involved in this embodiment contains VVD mutant 1 and VVD mutant 2, for example, in VVD mutant 1, the amino acids corresponding to positions 52 and 55 of the amino acid sequence represented by sequence number 1 can be arginine; in VVD mutant 2, the amino acid corresponding to position 52 of the amino acid sequence represented by sequence number 1 can be aspartic acid, and the amino acid corresponding to position 55 can be glycine. Alternatively, in VVD mutant 1, the amino acids corresponding to positions 16 and 19 of the amino acid sequence represented by sequence number 36 can be arginine; in VVD mutant 2, the amino acid corresponding to position 16 of the amino acid sequence represented by sequence number 36 can be aspartic acid, and the amino acid corresponding to position 19 can be glycine.

[0108] When the light-switching protein involved in this embodiment contains VVD mutant 1 and VVD mutant 2, in at least one of the mutants, the amino acid located in the FAD binding domain can be an amino acid different from the wild-type amino acid.

[0109] For example, in at least either VVD mutant 1 or VVD mutant 2, the amino acid corresponding to position 74 and / or position 85 of the amino acid sequence represented by sequence number 1 may be an amino acid different from the amino acid in the amino acid sequence represented by sequence number 1. Alternatively, in at least either VVD mutant 1 or VVD mutant 2, the amino acid corresponding to position 135 and / or position 165 of the amino acid sequence represented by sequence number 1 may be an amino acid different from the amino acid in the amino acid sequence represented by sequence number 1.

[0110] Alternatively, in at least one of the VVD mutants 1 or 2, the amino acid corresponding to position 38 and / or position 49 of the amino acid sequence represented by sequence number 36 may be an amino acid different from the amino acid in the amino acid sequence represented by sequence number 36. Alternatively, in at least one of the VVD mutants 1 or 2, the amino acid corresponding to position 99 and / or position 129 of the amino acid sequence represented by sequence number 36 may be an amino acid different from the amino acid in the amino acid sequence represented by sequence number 36.

[0111] More specifically, when the light-switching protein involved in this embodiment contains VVD mutant 1 and VVD mutant 2, for example, in VVD mutant 1 and VVD mutant 2, the amino acid corresponding to position 85 on the amino acid sequence represented by sequence number 1 can be valine, or the amino acids corresponding to positions 74 and 85 on the amino acid sequence represented by sequence number 1 can both be valine. Alternatively, when the light-switching protein involved in this embodiment contains VVD mutant 1 and VVD mutant 2, for example, in VVD mutant 1 and VVD mutant 2, the amino acids corresponding to positions 135 and 165 on the amino acid sequence represented by sequence number 1 can both be isoleucine.

[0112] Alternatively, when the light-switching protein involved in this embodiment includes VVD mutant 1 and VVD mutant 2, for example, in VVD mutant 1 and VVD mutant 2, the amino acid corresponding to position 49 on the amino acid sequence represented by sequence number 36 can be valine, or the amino acids corresponding to positions 38 and 49 on the amino acid sequence represented by sequence number 1 can both be valine. Alternatively, when the light-switching protein involved in this embodiment includes VVD mutant 1 and VVD mutant 2, for example, in VVD mutant 1 and VVD mutant 2, the amino acids corresponding to positions 99 and 129 on the amino acid sequence represented by sequence number 36 can both be isoleucine.

[0113] The two VVD proteins constituting the photo-switching protein of this embodiment can each bind to or fuse with "other substances (i.e., substances different from the proteins constituting the photo-switching protein)". This "other substance" is not particularly limited and can be any substance, such as proteins, lipids, carbohydrates, nucleic acids, and various other biological substances. The photo-switching protein of this embodiment can bind to the "other substance" through a connector or the like, and when the "other substance" is a protein, fusion is possible. More specifically, examples of "other substances" include... Figure 2 The "Cas9 Nter" and "Cas9 Cter" shown in the c are... Figure 7 The "CreN" and "CreC" shown in 'a' Figure 7 The "FlpN" and "FlpC" shown in c Figure 22 The "mVav2 N-ter" and "mVav2 C-ter" symbols shown in Figure a represent the interaction between "protein A" and "protein B". In this case, when the light-switching protein according to this embodiment is irradiated with blue light (for example, the wavelength region is not particularly limited, but for example, around 400 nm to 500 nm), the interaction between protein A and protein B can be induced, and when the blue light irradiation is stopped, protein A and protein B can dissociate. Therefore, by using the light-switching protein according to this embodiment, the interaction between proteins can be freely manipulated by switching light irradiation ON / OFF. In addition to the examples shown herein, the light-switching protein according to this embodiment has useful applications (see Non-Patent Document 1 and Non-Patent Document 2, etc.).

[0114] The second embodiment is a nucleic acid (DNA or RNA) encoding the light-switching protein involved in this embodiment.

[0115] The nucleic acid involved in this embodiment can be prepared by methods known in the art or by appropriately modifying those methods. For example, the desired nucleic acid can be obtained by introducing the desired mutation into the DNA encoding wild-type VVD. Alternatively, it can be synthesized using an automated synthesis apparatus. When the proteins constituting the light switch involved in this embodiment are fused with other proteins, the nucleic acid encoding such fusion protein is also included in the nucleic acid involved in this embodiment.

[0116] The light-switching protein involved in this embodiment can be prepared by: integrating the nucleic acid encoding it into a suitable expression vector, transforming or transfecting a suitable host cell with the expression vector, culturing it in a suitable culture medium, and purifying the expressed proteins.

[0117] As a host cell for protein expression, bacterial cells (e.g., Escherichia coli strain B , big Enterobacter K12 strain, Corynebacterium ammoniagenicum , Corynebacterium glutamicum , Liquefied Serratia , Streptomyces limonene , Pseudomonas putida etc.), mold (e.g., Penicillium campeporte , Cephalosporium (etc.), animal cells, plant cells, baculovirus / insect cells, or yeast cells (e.g., brewing yeast and Pichia pastoris etc.

[0118] Expression vectors used to express proteins can be adapted to various host cells. Examples of expression vectors include pBR322, pBR325, pUC118, pET (E. coli host), pEGFP-C, pEGFP-N (animal cell host), pVL1392, pVL1393 (insect cell host, baculovirus vector), pG-1, Yep13, or pPICZ (yeast cell host). These expression vectors have replication origins, selection markers, and promoters suitable for their respective vectors, and may include enhancers, transcription termination sequences (terminators), ribosome binding sites, and polyadenylation signals as needed. Furthermore, to facilitate the purification of the expressed peptide, base sequences for the fusion and expression of FLAG, His, HA, and GST tags can be inserted into the expression vector.

[0119] The expression vector can be prepared by methods known to those skilled in the art, or by using commercially available reagent kits, etc.

[0120] When extracting expressed proteins from cultured bacterial cells or cultured cells, after culturing, the bacterial cells or cultured cells can be collected using known methods, suspended in an appropriate buffer, and then the cells or cells can be disrupted using ultrasound, lysozyme, and / or freeze-thaw cycles. The soluble extract can then be obtained by centrifugation and filtration. Especially when using cultured cells as the host, it is preferable to obtain the protein expressed in the culture supernatant by recovering the supernatant. Known separation and purification methods can be appropriately combined to obtain the target protein from the obtained extract or culture supernatant. Commonly known separation and purification methods include: methods utilizing solubility such as salting out and solvent precipitation; methods primarily utilizing molecular weight differences such as dialysis, ultrafiltration, gel filtration, and SDS-PAGE; methods utilizing charge differences such as ion exchange chromatography; methods utilizing specific affinity such as affinity chromatography (e.g., using a resin with glutathione bound to the carrier when expressing a peptide with a GST tag, using Ni-NTA resin or Co-based resin when expressing a peptide with a His tag, using an anti-HA antibody resin when expressing a peptide with a HA tag, using an anti-FLAG antibody-bound resin when expressing a peptide with a FLAG tag); methods utilizing hydrophobicity differences such as reversed-phase high-performance liquid chromatography; and methods utilizing isoelectric point differences such as isoelectric focusing electrophoresis.

[0121] The third embodiment is a light-switching protein according to the first embodiment, in which two proteins selected from the VVD proteome are linked by a fluorescent protein or a bioluminescent protein. Depending on the situation, one or more amino acid residues at the terminal of the VVD protein that binds to the fluorescent or bioluminescent protein may be omitted (refer to the description regarding the first embodiment). The light-switching protein according to the third embodiment has a fluorescent or bioluminescent protein between the two VVD proteins. This fluorescent or bioluminescent protein may bind directly to the VVD protein or indirectly through a linker (e.g., a peptide).

[0122] When a fluorescent protein exists between two VVD proteins, if the fluorescent protein is excited with near-infrared light (e.g., two-photon excitation) that is permeable to biological tissues, and the excitation energy emitted by the fluorescent protein can be received by the VVD protein, FRET (Fluorescence Resonance Energy Transfer) will occur from the fluorescent protein to the VVD protein, inducing the VVD proteins to bind to each other. Two-photon excitation can be used to excite fluorescent proteins with near-infrared light. In two-photon excitation, half the energy of a single-photon excitation, i.e., two photons of twice the wavelength, are simultaneously absorbed by the fluorescent protein, thus producing fluorescence at the same wavelength as single-photon excitation. For example, if the fluorescent protein is excited with a wavelength of 400 nm in the case of single-photon excitation, then in the case of two-photon excitation, it becomes excited with a wavelength of 800 nm. In other words, using near-infrared light with a wavelength around 800 nm as the excitation light has the advantage of being less affected by scattering within biological samples (e.g., biological tissues), enabling deep observation. Therefore, the fluorescent protein constituting the light-switching protein according to the third embodiment is preferably a fluorescent protein capable of being two-photon excited by near-infrared light (e.g., light around 650 nm to 900 nm), and preferably a fluorescent protein that generates FRET from the VVD protein. Such a fluorescent protein can be easily selected by those skilled in the art, and is not particularly limited; examples include BFP, TagBFP, mTagBFP2, mBlueberry2, mCerulean, mTurquoise, ECFP, TagCFP, Rosmarius, meleCFP, mTFP1, KCY, and meffCFP.

[0123] Furthermore, when a bioluminescent protein exists between two VVD proteins, if the energy generated by the oxidation reaction catalyzed by the bioluminescent protein in the presence of a luminescent substrate can be received by the VVD protein, a BRET (Bioluminescence Resonance Energy Transfer) will occur from the bioluminescent protein to the VVD protein, inducing the binding of the VVD proteins to each other. Examples of bioluminescent proteins that can be used in this context include NanoLuc, Gaussian luciferase, Renalis luciferase, and firefly luciferase.

[0124] The fourth embodiment is a method for controlling the distance between two proteins, comprising: a step of binding (including "fusion") one VVD protein constituting the photoswitch protein of this embodiment to one of the two proteins; a step of binding (including "fusion") the other VVD protein constituting the photoswitch protein to the other of the two proteins; and a step of irradiating the photoswitch protein with light. Here, the two "proteins" are preferably interacting proteins. Furthermore, the two proteins can be two fragments resulting from the splitting of a single protein, and these fragments can be fragments split in a manner that restores the activity of the original protein upon rebinding (reassociation). The binding of the VVD protein to the protein can be via a linker or the like, or it can be a direct binding (fusion).

[0125] The fourth embodiment can also be implemented intracellularly. Here, "cell" includes both in vitro cells and in vivo cells. The method for introducing the protein complex consisting of the light-switching protein according to this embodiment and the "two proteins" according to the fourth embodiment into the cell can be appropriately selected by those skilled in the art. For example, a vector expressing the protein complex can be introduced into the cell to express the protein complex within the cell, or a cell membrane-permeable peptide can be used to directly introduce the protein complex into the cell. In the case of migrating the protein complex to the nucleus or intracellular organelles, it can be introduced into the cell in a state where the protein complex is fused with a migration signal peptide (nuclear localization signal peptide, mitochondrial localization signal peptide, etc.).

[0126] When this instruction manual is translated into English, the singular pronouns “a,” “an,” and “the” will be used in both singular and plural forms unless the context explicitly states otherwise. Furthermore, in this instruction manual, “equal degree” and “approximately” refer to a numerical range of ±10%.

[0127] The following embodiments are shown to further illustrate the present invention, but these embodiments are merely examples of implementation of the present invention and do not limit the scope of the present invention.

[0128] Example

[0129] 1. Methods

[0130] 1-1. Reagents and Materials

[0131] All oligonucleotides used in plasmid construction were synthesized by Eurofines Genomics, Integrated DNA Technology, or GenScript. The mammalian cell expression vectors pcDNA3.1(+), pcDNA3.1 / V5-His A, and pEF6 / V5-His A were purchased from ThermoFisher Scientific. Plasmid construction was performed using existing methods based on overlap extension PCR, restriction enzyme digestion of PCR products, and T4 DNA ligase ligation of double-stranded DNA. For PCR reactions, PrimeSTAR HS DNA polymerase (TakaraBio, Japan) or KOD One PCR Master Mix (TOYOBO) were used according to the accompanying instructions. All restriction enzymes used for DNA digestion were purchased from Takara Bio or New England BioLabs (NEB) and used according to the accompanying instructions. The T4 DNA ligase used for the ligation reaction was Ligation high Ver.2 (TOYOBO) according to the accompanying instructions. All constructed constructs were validated using a DNA sequencing analysis service (Eurofines Genomics). All plasmid constructs were purified using QIAGEN Plasmid Plus Kits (QIAGEN).

[0132] 1-2. Plasmid Construction

[0133] To construct an expression plasmid for photoactivated Cas9, the expression plasmid fused with the nuclear localization signal (NLS) from the SV40 T antigen was optimized. Streptococcus pyogenesThe coding sequences for the N-terminal and C-terminal segments of the Cas9 codons were amplified from Addgene plasmid (ID: 42230). The coding sequences for pMag and nMagHigh1 codons, optimized for expression in mammalian cells, were commissioned to Eurofines Genomics, Integrated DNA Technology, or GenScript for synthesis. The base sequences of expression constructs for PA-Cas9_N-term, PA-Cas9_C-term, scPA-Cas9 (ligated with a flexible adapter), scPA-Cas9 (ligated with a rigid adapter), and scPA-Cas9 (direct fusion) were inserted into the HindIII and XhoI sites of the pcDNA3.1 / V5-His A vector containing the cytomegalovirus (CMV) promoter. The gRNA expression vector was prepared by introducing annealed oligoDNA into the BbsI site of the pSPgRNA vector (ID: 47108) purchased from Addgene. Human VEGFA ,people DNMT1 ,people EMX1 and mice K-ras The target gRNA. Target sequences and oligonucleotides used to construct gRNA expression vectors, such as... Figure 4 As shown in Table 1.

[0134] [Table 1]

[0135]

[0136] To construct photoactivated Cre, the N-terminal and C-terminal fragments of the Cre recombinase, optimized for expression in mammalian cells, were fused with the NLS (nuclear localization signal) sequence and commissioned to Invitrogen or ThermoFisher Scientific for synthesis. The coding sequences for pMag (SEQ ID NO: 37) and nMag (SEQ ID NO: 38), optimized for expression in mammalian cells, were commissioned to Eurofines Genomics, Integrated DNA Technology, or GenScript for synthesis. The photoactivated Cre sequence was constructed by inserting PA-Cre and scPA-Cre into the HindIII-XbaI or HindIII-XhoI site of the pcDNA3.1(+) vector containing the CMV promoter. Regarding the use of Cre- for detection... loxP Construction of a luciferase reporter gene with recombinase activity, in conjunction with loxPAdjacent polyadenylation "termination" signal (poly(A)) repeat sequences at the same locus were amplified from Addgene plasmid (ID: 22797). The sequence encoding firefly luciferase (Fluc) was amplified from the pGL4.31 vector (Promega). loxP -stop- loxP - The Fluc construct sequence is inserted into the HindIII-XhoI site of the pcDNA3.1(+) vector with the CMV promoter.

[0137] To construct the photoactivated Flp, the N-terminal and C-terminal fragments of the Flp recombinase, optimized for expression in mammalian cells, were fused with NLS and synthesized by Eurofines Genomics. The coding sequences for pMag, nMag, pMagHigh1, and nMagHigh1, optimized for expression in mammalian cells, were synthesized by Eurofines Genomics, Integrated DNA Technology, or GenScript. The photoactivated Flp sequence was constructed by inserting the PA-Flp_N-terminal, PA-Flp_C-terminal, and scPA-Flp sequences into the HindIII-XbaI site of the pcDNA3.1(+) vector with a CMV promoter. The sequence was used to detect Flp- FRT A luciferase reporter gene construct with recombinase activity is created by... loxP -stop- loxP -Two Fluc constructs loxP The sites are respectively used FRT The site substitution was used to create the photoactivated Flp construct associated with Cre-dependent recombination cascades by inserting the scPA-Flp sequence into... loxP -stop- loxP The Fluc reporter gene vector was constructed using the HindIII-NotI site. It is used to detect Flp- FRT The fluorescent reporter gene with recombinase activity was synthesized by entrusting Eurofines Genomics with the synthesis of a sequence encoding mKate2 fused with Akaluc luciferase, and the synthesized sequence was inserted into... FRT -stop- FRT - The vector for the Fluc reporter gene was constructed using the HindIII-NotI site.

[0138] To construct photoactivated Vav, the coding sequences for Lifeact-mCherry (codons optimized for expression in mammalian cells), the P2A self-cleaving 2A peptide, the N-terminal and C-terminal fragments of mouse Vav2, Rho GEF, pMag, and nMag were commissioned to Eurofines Genomics for synthesis. The synthesized sequences were inserted into the EcoRI-XbaI site of the pEF6 / V5-His A vector with a human EF-1α promoter. For the construction of two-photon excited photoactivated Vav, the sequences encoding pMag, mTagBFP2, and nMag were commissioned to Eurofines Genomics for synthesis and inserted into the AgeI-KpnI site of the scPA-Vav construct.

[0139] The complete base sequence of the constructed construct is shown below. Figures 28-43 .

[0140] 1-2. Cell Culture

[0141] HEK293T cells, COS-7 cells, NIH3T3 cells (American Type Culture Collection (ATCC)) and HeLa cells (Japanese Collection of Research Bioresources (JCRB) cell bank) were cultured in Dulbecco's Modified Eagle Medium (Thermo Fisher Scientific) supplemented with 0% fetal bovine serum (Thermo Fisher Scientific), 100 units / mL penicillin and 100 μg / mL streptomycin (Thermo Fisher Scientific), and 2 mM L-glutamine or 2 mM GlutaMAX (Thermo Fisher Scientific) at 37°C under 5% CO2 conditions.

[0142] From LSL-KrasG12D / +; LSL-Trp53R172H / +; ptf1a-CreThe 5-11 cell line of pancreatic cancer from (KPC) mice was established according to a previously reported method (Fuchigami et al., Scientific Reports 8:14211 (2018)). The 5-11 cells were cultured in RPMI-1640 medium (Roswell Park Memorial Institute 1640 Medium) (Thermo Fisher Scientific) supplemented with 20% FBS and 1× nacalai tesque at 37°C under 5% CO2 conditions.

[0143] 1-3. Light source

[0144] For the blue light used in the detection of cultured cells and in vivo mouse experiments, an LED light source (70±20nm; CCS) was used.

[0145] 1-4. Genome Editing Experiments

[0146] For the InDel mutation assay using HEK293K cells via non-homologous end joining (NHEJ), cells were seeded at 100,000 cells / well in 24-well plates (Thermo Fisher Scientific) and cultured at 37°C for 24 hours under 5% CO2. Subsequently, transfection was performed using Lipofectamine 3000 (Thermo Fisher Scientific) according to the accompanying instructions. The cDNA encoding the NLS-N-terminal fragment of Cas9-pMag, the nMagHigh1-C-terminal fragment of Cas9-NLS, and gRNA were in a 1:1:1 ratio. The total cDNA amount was 0.2741 μg / well. As positive and negative controls, cDNA encoding full-length Cas9 or pcDNA3.1 / V5-His A (empty vector) was transfected at a 1:1 ratio. Twenty hours after transfection, cells were in the dark or under blue light (1.0 Wm). -2 The cells were cultured for 24 hours. Afterward, the genomic DNA was recovered using the Blood Cultured Cell Genomic DNA Extraction Mini Kit (Favorgen) according to the accompanying instructions.

[0147] For the InDel mutation assay using HeLa cells via non-homologous end ligation, cells were seeded at a rate of 100,000 cells / well in 24-well plates (Thermo Fisher Scientific) and cultured at 37°C for 96 hours under 5% CO2. Subsequently, transfection was performed using X-tremeGENE 9 reagent (Thermo Fisher Scientific) according to the accompanying instructions. The cDNA encoding the NLS-N-terminal fragment of Cas9-pMag, the nMagHigh1-C-terminal fragment of Cas9-NLS, and gRNA was in a 1:1:1 ratio. The cDNA encoding scPA-Cas9 and gRNA was in a 1:1 ratio. The total cDNA amount was 0.1 μg / well. As positive and negative controls, cDNA encoding full-length Cas9 or pcDNA3.1 / V5-HisA (empty vector) was transfected at a 1:1 ratio. Immediately after transfection, cells were placed in the dark or under blue light (1.0 Wm). -2 The cells were cultured for 48 hours. Afterward, the genomic DNA was recovered using the QuickExtract DNA Solution kit (Lucigen) according to the accompanying instructions.

[0148] For the InDel mutation experiment using 5-11 cells via non-homologous end ligation, cells were seeded at 100,000 cells / well in 10cm culture dishes and cultured at 37°C for 24 hours under 5% CO2 conditions. Next, gene transfer was performed at 400,000 cells / cuvette using the SG Cell Line 4D-Nucleofector X kit S (Lonza) and EN-158 program, following the accompanying instructions. cDNA encoding the NLS-N-terminal fragment of Cas9-pMag, the nMagHigh1-C-terminal fragment of Cas9-NLS, and gRNA were transfected at a 1:1:1 ratio. CDNA encoding scPA-Cas9 and gRNA was transfected at a 2:1 ratio. The total cDNA volume was 2.0 μg / well. As positive and negative controls, cDNA encoding full-length Cas9 or pcDNA3.1 / V5-His A (empty vector) was transfected nuclearly at a ratio of 2:1. After nuclear transfection, cells were incubated in Nucleocuvette at room temperature for 10 minutes. Subsequently, cells were suspended in preheated RPMI-1640 medium. The suspended cells were seeded at a rate of 40,000 cells / well in 96-well plates, and immediately after seeding, the cells were incubated in the dark or under blue light (1.0 Wm). -2 The cells were cultured for 48 hours. Afterward, the genomic DNA was recovered.

[0149] 1-5. Genomic PCR

[0150] Genomic regions containing nuclease target sites were amplified using PrimeSTAR HS DNA polymerase (TaKaRa Bio) via decremental PCR. Decremental PCR was performed under the following conditions.

[0151] • Initial denaturation; 98℃ for 3 minutes,

[0152] • Decreasing PCR (10 cycles);

[0153] Denaturation (98℃, 10 minutes), annealing (starting from 72℃, decreasing by 1℃ for 30 seconds per cycle), extension (72℃, 1 minute).

[0154] • Typical PCR (25 cycles);

[0155] Denaturation (98℃, 10 seconds), Annealing (62℃, 30 seconds), Extension (72℃, 1 minute)

[0156] The final extension reaction was carried out at 72°C for 3 minutes.

[0157] The sequences of the PCR primers are shown in Table 2. The amplified PCR products were purified using the FastGene Gel / PCR Extraction Kit (Nippon Genetics, Japan).

[0158] [Table 2]

[0159]

[0160] 1-6. Sequencing Analysis

[0161] The PCR product prepared as described above was subcloned into pcDNA3.1 / V5-His A. The plasmid was isolated and Sanger sequencing was performed. TIDE resolution (https: / / tide.deskgen.com / ) was performed using the same purified PCR product.

[0162] 1-7. Site-Specific Recombination Experiment

[0163] For luciferase detection, COS-7 cells or NIH3T3 cells were seeded at 10,000 cells / well or 15,000 cells / well into 96-well clear flat-bottomed black-walled microplates (Greiner Bio-One, Austria) and cultured at 37°C for 24 hours under 5% CO2 conditions. Using Lipofectamine 3000 reagent, cDNA encoding PA-Cre, scPA-Cre, PA-Flp, or scPA-Flp and the luciferase reporter gene were analyzed at 37°C. loxP -stop- loxP Fluc reporter gene or FRT -stop- FRT The Fluc reporter gene was transfected into cells at a ratio of 1:20. The total amount of DNA used was 0.1 μg / well. 24 hours after transfection, the cells were incubated in the dark or under blue light (1.0 Wm²). -2 Incubate for 24 hours. Just before performing the luciferase assay, replace the medium with HBSS (Hanks' balanced salt solution) containing 0.2 mM D-luciferin potassium salt (Wako) (Thermo Fisher Scientific). For bioluminescence determination, use Centro XS at room temperature. 3 The LB 960 microplate luminescence detector (Berthold Technologies) measures each well for 10 seconds.

[0164] about Figure 9 and Figure 11 The control experiment shown involved seeding COS-7 cells at 10,000 cells / well or 15,000 cells / well into 96-well clear, flat-bottomed, black-walled microplates and culturing at 37°C for 24 hours under 5% CO2 conditions. The cDNA encoding PA-Cre or scPA-Cre was then inoculated using X-tremeGENE 9 reagent at 37°C. loxP -stop- loxP The Fluc reporter gene was transfected into cells at a ratio of 1:10. The total amount of DNA used was 0.05 μg / well. The cDNA encoding PA-Flp or scPA-Flp was transfected into cells using Lipofectamine 3000 reagent at 37°C. FRT -stop- FRT The Fluc reporter gene was transfected into cells at a ratio of 1:10. The total amount of DNA used was 0.1 μg / well. 24 hours after transfection, the cells were incubated in the dark or under blue light (1.0 Wm²). -2Incubate for 24 hours. Just before performing the luciferase assay, replace the medium with HBSS medium containing 0.2 mM D-luciferin potassium salt. For bioluminescence determination, use Centro XS at room temperature. 3 LB 960 microplate luminescence detector, measuring each well for 10 seconds.

[0165] about Figure 9 and Figure 11 The irradiation intensity-dependent experiment shown involved seeding COS-7 cells at 10,000 cells / well or 15,000 cells / well into 96-well clear, flat-bottomed, black-walled microplates and culturing at 37°C for 24 hours under 5% CO2 conditions. The cDNA encoding scPA-Cre was then inoculated using X-tremeGENE 9 reagent at 37°C. loxP -stop- loxP The Fluc reporter gene was transfected into cells at a ratio of 1:10. The total amount of DNA used was 0.05 μg / well. The cDNA encoding scPA-Flp and... were transfected using Lipofectamine 3000 reagent at 37°C. FRT -stop- FRT The Fluc reporter gene was transfected into cells at a ratio of 1:10. The total amount of DNA used was 0.1 μg / well. Twenty-four hours after transfection, cells were in the dark or under blue light (0.01, 0.03, 0.1, 0.3, 1.0, 1.6, and 3.3 Wm). -2 Or 0.01, 0.03, 0.05, 0.1, 0.3, 1.0, 1.6 and 3.0Wm -2 Incubate for 24 hours. Just before performing the luciferase assay, replace the medium with HBSS medium containing 0.2 mM D-luciferin potassium salt. For bioluminescence determination, use Centro XS at room temperature. 3 LB960 microplate luminescence detector (Berthold Technologies, Germany), 10 seconds per well.

[0166] about Figure 9 and Figure 11 The irradiation time-dependent experiment shown involved seeding COS-7 cells at 10,000 or 15,000 cells / well into 96-well clear, flat-bottomed, black-walled microplates and culturing at 37°C for 24 hours under 5% CO2 conditions. The cDNA encoding scPA-Cre was then inoculated using X-tremeGENE 9 reagent at 37°C. loxP -stop- loxPThe Fluc reporter gene was transfected into cells at a ratio of 1:10. The total amount of DNA used was 0.05 μg / well. The cDNA encoding scPA-Flp and... were transfected using Lipofectamine 3000 reagent at 37°C. FRT -stop- FRT The Fluc reporter gene was transfected into cells at a ratio of 1:10. The total amount of DNA used was 0.1 μg / well. Twenty-four hours after transfection, the cells were exposed to blue light (1.0 Wm²) for 30 seconds, 3 minutes, 30 minutes, and 1 hour in the dark. -2 Afterwards, they are cultured in the dark or exposed to 24 hours of blue light (1.0Wm). -2 Before performing the luciferase assay, replace the medium with HBSS medium containing 0.2 mM D-luciferin potassium salt. For the bioluminescence assay, use Centro XS at room temperature. 3 LB 960 microplate luminescence detector (Berthold Technologies, Germany), 10 seconds per well.

[0167] about Figure 15 The recombinant cascade experiment shown involved seeding COS-7 cells at 15,000 cells / well into 96-well clear, flat-bottomed, black-walled microplates and culturing at 37°C for 24 hours under 5% CO2 conditions. Using Lipofectamine 3000 reagent at 37°C, cDNA encoding iCre or pcDNA3.1(+) empty vector, cDNA encoding scPA-Flp, and the Akaluc luciferase reporter gene were transfected into the cells at a ratio of 1:1:9. The total amount of DNA used was 0.1 μg / well. Using Lipofectamine 3000 reagent at 37°C, cDNA encoding PA-Flp or scPA-Flp and... FRT -stop- FRT The Fluc reporter gene was transfected into cells at a ratio of 1:10. The total amount of DNA used was 0.1 μg / well. 24 hours after transfection, the cells were incubated in the dark or under blue light (1.0 Wm²). -2 Incubate for 24 hours. Just before the luciferase assay, change the medium to HBSS medium containing 0.2 mM D-luciferin potassium salt. For bioluminescence determination, use Centro XS at room temperature. 3 LB 960 microplate luminescence detector, measuring each well for 10 seconds.

[0168] 1-8. In vivo experiments using mice

[0169] Experiments using mice were conducted in accordance with guidelines concerning the management and use of laboratory animals. Six-week-old females were used in all experiments. Rosa26 - Believe T2 mice (CreER) T2 Female mice (provided by Dr. Tyler Jacks of MIT). Hair was removed from the ventral surface using depilatory cream. The depilated mice were allowed to rest in their cages for at least 12 hours. Six hours prior to the injection of plasmid cDNA into the HTV (hydrodynamic tail vein), in order to activate CreER... T2 The recombinase was administered intraperitoneally to mice via injection of tamoxifen (Sigma) at a dose of 100 μg / body weight (g). Next, the enzyme encoding pcDNA3.1- loxP -stop- loxP -scPA-Flp cDNA and encoding pcDNA3.1- FRT stop- FRT -Akaluc-mKate2 cDNA was injected into mice via HTV at a 1:1 ratio using TransIT-QR Hydrodynamic Delivery Solution (Mirus Bio LLC), according to the accompanying instructions. The total amount of DNA injected was 40 μg / mouse. The total amount of solution injected was 0.1 mL / mouse. After DNA injection, mice were housed in the dark for 6 hours, and then exposed to an LED light source (470 ± 20 nm; 132 Wm). -2 The mice were exposed to light for 18 hours. Fluorescence imaging was performed on the mice 24 hours after DNA injection. The livers of the DNA-injected mice were isolated using standard surgical methods. The isolated livers were then immersed in PBS solution. Fluorescence images of the isolated livers were then obtained using an integrated fluorescence microscope BZ-X710 (KEYENCE).

[0170] 1-9. Imaging of live cells

[0171] Glass dishes (Cat.#D11530H, Matsunami) were treated with 25 mg / mL human fibronectin (BDBiosciences) at room temperature for 10 minutes, followed by two washes with 2 mL Mili-Q water. For cell migration detection via confocal imaging, NIH3T3 cells were seeded at 2000 cells / dish on 35 mm glass dishes (glass area = φ14 mm) and cultured at 37°C for 24 hours under 5% CO2. Using Lipofectamine 3000 reagent, cDNA encoding Lifeact mCherry and scPA-Vav or 2PscPA-Vav was transfected into the cells at 37°C. The total amount of transfected DNA was 0.1 μg / dish. Twenty-four hours after transfection, the culture medium was changed to Leibovitz's L-15 phenol red-free medium containing 5% FBS and 2 mM GlutaMAX. Imaging was performed using a 63 / 1.05 numerical aperture (NA) silicone oil immersion objective (Carl Zeiss) on an LSM confocal laser scanning microscope (Carl Zeiss) stage equipped with a heated stage adapter (Tokai Hit). Fluorescence images of mCherry were acquired using a multi-line helium-neon laser (543 nm). Single-photon blue light illumination was performed using a 1 mW multi-line argon laser (488 nm). Two-photon excitation of mTagBFP2 was performed using a 25 mW multi-line CHAMELEON laser (808 nm).

[0172] 1-10. Quantitative analysis of cellular region stretching

[0173] Fluorescence images of NIH3T3 cells expressing Lifeact-mCherry and scPA-Vav or 2PscPA-Vav were acquired 15 minutes before light exposure (dark), just before light exposure (before), and 15 minutes after light exposure (after). The acquired fluorescence images were converted to binary data based on the fluorescence intensity of mCherry using Image-J software. The cell region stretching percentage (%) between (dark) and (before) or (before) and (after) was calculated using the obtained data.

[0174] 1-11. Reproducibility and Statistical Analysis

[0175] No detectability tests were performed in the cell culture and mouse experiments. No samples or animals were excluded from the analysis. Randomization was performed in the cell culture and mouse experiments. Blinding was not performed in the cell culture and mouse experiments.

[0176] 2. Results

[0177] 2-1. Development of scMagnet

[0178] Based on the Cas9 endonuclease and magnet system (Kawano et al., Nat. Commun. 6:6256 (2015)), the inventors have developed a bimolecular type of photoactivated Cas9 (PA-Cas9; photoactivatable Cas9) useful for optogenetic genome editing (Nihongaki et al., Nat. Biotechnol. 33:755-760 (2015)) (see reference). Figure 2 a, Figure 3 and Figure 4 First, the photoinducibility of existing PA-Cas9 activity was determined by tracking of indels by decomposition (TIDE) (Brinkman et al., Nucleic Acids Res. 42: e168 2014). Under blue light irradiation, expression of human... VEGFA HEK293T cells targeting PA-Cas9 showed an equally high InDel mutation introduction rate of over 25% as cells expressing full-length Cas9. Figure 1 (b). Additionally, PA-Cas9 is effective for human cells within HEK293T cells. DNMT1 Genes and EMX1 The gene also efficiently introduced the InDel mutation in a light-dependent manner. Figure 5 ).

[0179] On the other hand, expressing people VEGFA HeLa cells targeting the gene PA-Cas9 showed a very low InDel mutation introduction rate of less than 1% under both dark and blue light irradiation conditions. Figure 2 (d). Furthermore, with people VEGFA Similarly, in cases where genes are targeted, expression is also targeted at humans. DNMT1 and EMX1 HeLa cells targeting the gene PA-Cas9 also exhibited a low InDel mutation introduction rate almost identical to that of cells expressing the empty pcDNA3.1 vector. Figure 5 Generally speaking, it is known that the expression level of exogenous proteins in HeLa cells is low compared with HEK293T cells (Rio et al., Science 227:23-28 (1985)). Therefore, it is believed that the low light-induced efficiency of PA-Cas9 in HeLa cells is due to the low expression level of PA-Cas9 in the cells.

[0180] To address the problem in existing bimolecular optical switching systems (e.g., those composed of pMag and nMag bimolecules in existing magnetic systems), namely the dependence of photoinduction efficiency on the intracellular concentration of magnet proteins, such as... Figure 2 As shown in Figure c, the pMag and nMag are considered to be connected in series. Thus, the substance formed by connecting bimolecular magnet proteins in series is called scMagnet (single-chain-based Magnet).

[0181] The fragments of Cas9 were combined with pMag and nMag respectively to create scPA-Cas9 (single-chain-based photoactivatable Cas9) by connecting them in series. Figure 2 (c) The photoinducibility efficiency of this genome editing (the introduction of the InDel mutation into the target genomic region) was measured. The results, as expected, showed that in the expression of human... VEGFA In HeLa cells targeting the gene scPA-Cas9, to VEGFA The gene region was introduced with a high InDel mutation rate of over 10%. Figure 2 (d). On the other hand, in the dark, it exhibits a low InDel frequency of about 1% to 4%, similar to that of the pcDNA3.1 empty vector. Based on this result, the photoinduction efficiency of scPS-Cas9 activity in HeLa cells is about 18.1 times that of PA-Cas9 activity, indicating a significant improvement. The characteristics of scPA-Cas9, which exhibits high genome editing efficiency under blue light irradiation and almost no genome editing in the dark, are significant for other genes (human) in HeLa cells. DNMT1 and EMX1 The same applies when the target is a gene. Figure 6 ).

[0182] The above results demonstrate that scMagnet functions as a single-molecule type of optical switch system with the unique characteristics of low leakage activity (binding activity in the dark) and extremely high recombination induction rate under blue light irradiation.

[0183] 2-2. Verification of the in vitro effects of scMagnet

[0184] 2-2-1. The versatility of scMagnet

[0185] Next, we will investigate whether the scMagnet system can be applied to the optogenetic rebinding of other split proteins besides Cas9. To date, the inventors have developed photoactivated Cre recombinase (PA-Cre) (Kawano et al., Nat. Chem. Biol. 12:1059-1064 (2016)) and photoactivated Flp recombinase (PA-Cre) (Jung et al., Nat. Commun. 10:314 (2019)) based on the Magnet system. Therefore, to study the versatility of the scMagnet system, we will fabricate single-stranded photoactivated Cre recombinase and single-stranded photoactivated Flp recombinase based on scMagnet and study their activities (…). Figure 7 a and c Figure 8 , Figure 10 First, the photoinduction efficiency of scPA-Cre or scPA-Flp and that of A-Cre or PA-Flp were compared using luciferase reporter gene assays in COS-7 cells. The results showed that scPA-Cre, like PA-Cre, exhibited high photoinducible recombination activity (up to 122-fold) under continuous light irradiation, short-duration pulsed irradiation, and light irradiation of different intensities, but did not exhibit leakage activity in the dark. Figure 9 In addition, scPA-Flp, like PA-Flp, exhibited highly efficient photoinduced recombination activity (up to 122-fold) and did not show leakage activity in the dark. Figure 11 Based on the above results, it is shown that not only the Cas9 endonuclease, but also scMagnet can induce optogenetic rebinding of other segmentation proteins, including the Cre recombinase and the Flp recombinase.

[0186] Next, we investigated whether suppressing the expression levels of scPA-Cre and PA-Cre in COS-7 cells affected the recombination efficiency induced by scPA-Cre and PA-Cre. The transfection amounts of plasmid DNA encoding scPA-Cre and plasmid DNA encoding PA-Cre were reduced from 9.1% of the total transfection amount. Figure 9 The experimental conditions shown in the data indicate that the expression level decreased to 4.8%. Under these conditions, even with decreased expression, scPA-Cre exhibited higher photoinduced recombination activity than PA-Cre under blue light irradiation. Figure 7 (b). The same result was observed when using the Flp recombinase for splitting ( Figure 7 d). Furthermore, the same results were obtained when using NIH3T3 cells with lower expression levels of the exogenous gene protein compared to COS-7 cells. Figure 12 ).

[0187] The results above demonstrate that the scMagnet system can effectively control the optogenetic recombination of split proteins even in cells with low expression levels of proteins from exogenous genes.

[0188] 2-2-2. Effects of linker length or amino acid deficiency at the pMag-nMag junction on activity.

[0189] This study investigated the effect of the linker length connecting pMag and nMag on the photoinduction efficiency of the scMagnet system. scPA-Cre was fabricated by connecting pMag and nMag using flexible peptide linkers (linkers composed of 0–20 amino acid residues). Figure 13 a). The photo-induced efficiency of recombination activity of scPA-Cre without connectors and scPA-Cre with flexible connectors was compared ( Figure 13 (a). "iCre" is a full-length Cre linked to a nuclear transfer signal and is a control that does not respond to blue light. "Reporter only" is a control where neither scPA-Cre nor iCre is used, only the bioluminescent reporter gene is present. The experimental results showed that, regardless of the adapter length, recombination activity was observed in all scPA-Cre with the inserted flexible adapter at a photoinduction efficiency comparable to that of scPA-Cre without the adapter. Figure 13 a). The characteristics of such scMagnet systems can also be confirmed when using Cas9 for genome editing. Figure 14 ).

[0190] Next, it was found that the leakage activity of scPA-Cre (without a linker) was further inhibited when several amino acid residues at the C-terminus of pMag were missing. Figure 13 (b). It should be noted that, since there is a trend that the photoinduction efficiency of recombination decreases when amino acid residues on the C-terminal side of pMag are missing, the number of missing amino acids is not particularly limited, but is preferably around 6.

[0191] The effect of deleting several amino acid residues at the N-terminus of nMag on its activity was further investigated. The enzyme activity of scPA-Cre, a monomolecule formed by directly linking the C-terminus of pMag to the N-terminus of nMag, was measured under both dark and blue light irradiation. The enzyme activity of scPA-Cre mutants (Δ1–Δ19) with amino acid deletions one by one at the N-terminus of nMag was also measured. Figure 15 The amino acids surrounding the junction of pMag and nMag are shown in [the diagram]. Figure 15In the control group, “iCre” is a full-length Cre gene linked to a nuclear transfer signal and does not respond to blue light. “reporter only” is a control group that does not use either scPA-Cre or iCre and only uses a bioluminescent reporter gene.

[0192] The results show that the scMagnet mutants obtained by deleting one residue at a time from the 1st to the 19th amino acid position of nMag have low leakage activity in the dark but maintain high responsiveness under blue light.

[0193] The Cre recombination activity was investigated when further staged deletion of amino acids on the N-terminal side of nMag was achieved. Figure 16 The amino acid sequence and domain structure of nMag are shown in [the diagram]. Figure 16 A. The nMag mutant, obtained by deleting amino acids with sequence numbers smaller than Δ as indicated in the figure, was introduced to prepare the scPA-Cre mutant, and its Cre recombination activity was evaluated. Figure 16 (B). Additionally, a scPA-Cre mutant (pMag+nMag-Latch-NCap-Hinge) containing only the nMag mutant with Latch-NCap-Hinge was also prepared to evaluate its Cre recombination activity. The enzyme activities of single-stranded photoactivated Cre recombinase (scPA-Cre, pMag+nMag) with N-terminal and C-terminal fragments of split-Cre introduced at both ends of the scMagnet, respectively, were measured under darkness and blue light. The enzyme activities of scPA-Cre mutants (Δ35, Δ56, Δ71, or Δ119) with a significant deletion of the N-terminus of nMag, the scPA-Cre mutant (Latch-NCap-Hinge) with nMag introduced only at the N-terminus, and the scPA-Cre mutant (ΔnMag) with only pMag introduced and no nMag introduced are shown in the figure. Figure 16 B. "iCre" is a full-length Cre linked to a nuclear transfer signal and is a control that does not respond to blue light. "reporter only" is a control in which neither scPA-Cre nor iCre is used, and only a bioluminescent reporter gene is used.

[0194] The results showed that even with the deletion of amino acids up to position 35 of nMag, the leakage activity of the scMagnet mutant was low in the dark. However, if amino acids up to position 56 of nMag were deleted, the leakage activity of the scMagnet mutant increased significantly in the dark. Based on these results, it is believed that the leakage activity of scPA-Cre, which introduces an nMag mutant with deletions of amino acid residues on the N-terminal side, changes from "low" to "high" depending on the deletion of any amino acid residue from the N-terminal side of nMag up to positions 36 to 55.

[0195] Next, the enzyme activity of scPA-Cre and the Cre recombination activity of scPA-Cre mutants containing mutants with partial deletions of the C-terminus of pMag and the N-terminus of nMag were measured under darkness and blue light irradiation. Figure 17 “iCre” is a full-length Cre linked to a nuclear transfer signal and is a control that does not respond to blue light. “reporter only” is a control in which neither scPA-Cre nor iCre is used and only a bioluminescent reporter gene is used.

[0196] Depend on Figure 17 It is known that by linking the pMag mutant with the amino acid residues missing on the C-terminal side to the nMag mutant with the amino acid residues missing on the N-terminal side, a scMagnet mutant with low leakage activity in the dark and high responsiveness (recombination activity) under blue light irradiation can be obtained.

[0197] 2-3. Verification of the effects of scMagnet in vivo

[0198] Next, the in vivo effects of the scMagnet system were verified using scPA-Flp. First, a recombination cascade system was created based on photo-induced recombination of scPA-Flp induced by Cre recombination. Figure 18 (a). First, using this system, it was confirmed in vitro that in COS-7 cells, not only blue light irradiation, but also Cre expression could efficiently induce scPA-Flp recombination (a). Figure 18 (b). Next, in order to operate this recombinant cascade system in vivo, locally expressed tamoxifen-induced Cre recombinase was used. Rosa26 Runtime CreER T2 The experiment was conducted using a mouse model (Ventura et al., Nature 445:661-665 (2007)). The plasmid DNA encoding fluxed-stop scPA-Flp and the plasmid encoding... were administered via hydrodynamic tail vein injection (Liu et al., Gene Ther. 6:1258-1266 (1999)). FRT Plasmid DNA of the floxed-stop mKate2 reporter gene was introduced into the liver of mice. Figure 19 a and b, Figure 20 Mice injected with DNA were housed in the dark or under blue light for 18 hours. When housed under blue light, mKate2 fluorescence was detected in the livers of mice injected with tamoxifen. Figure 19 c Figure 20 In contrast, no fluorescence was detected in the livers of mice raised in the dark. Figure 19 c Figure 20 Regarding fluorescence, mKate2 fluorescence was not detected in mice that had never been injected with tamoxifen and were fed in the dark and under blue light. These results indicate that scPA-Flp functions well in vivo in the presence of Cre and blue light. Figure 19 c Figure 20 ).

[0199] The above results demonstrate that scMagnet can also function in vivo through non-invasive blue light irradiation.

[0200] 2-4. Research on the reversibility of scMagnet's function

[0201] Using the Rho-family guanine exchange factor (GEF) Vav2 protein, which is required for cell stretching, this study investigated whether scMagnet can reversibly function in dependence on blue light irradiation. Figure 21 ).

[0202] First, scMagnet is fabricated using photo-activated Vav2 (called scPA-Vav). Figure 22 (a, b). Lifeact-mCherry was used as a fluorescent biosensor for detecting actin polymerization. The activity of scPA-Vav before and after blue light irradiation was evaluated by cell area expansion assay. Figure 23 ).

[0203] NIH3T3 cells expressing scPA-Vav showed almost no change in cell area after 15 minutes in the dark, but significant stretching of cell area was induced after 15 minutes of blue light irradiation. Figure 22 c and d, Figure 24 On the other hand, in the presence of EHop-016, which inhibits the binding of Vav2 to Racl1, no stretching of the cellular region was observed in the same manner as when the cells were maintained in the dark in the absence of EHop-016. Figure 22 e and f, Figure 24 The above results indicate that scPA-Vav does not exhibit leakage activity in the dark, but rather relies on blue light irradiation to effectively activate Vav2 signal transduction.

[0204] Next, to investigate whether scPA-Vav could reversibly function, scPA-Vav-expressing cells were irradiated with blue light and then kept in the dark. The results showed that scPA-Vav induced stretching of cell regions after 15 minutes of blue light irradiation, but the stretched regions disappeared when the cells were kept in the dark for 15 minutes. Figure 22 g). This result demonstrates that scMagnet can reversibly control the binding and dissociation of split proteins.

[0205] 2-5. Control of the scMagnet system based on two-photon excitation

[0206] When enabling the scMagnet system to function within biological tissues, it is believed that using two-photon excitation with long-wavelength near-infrared light, compared to single-photon excitation, allows for more efficient activation of scMagnet at deeper tissue depths. Furthermore, two-photon excitation microscopy offers higher spatiotemporal resolution compared to single-photon excitation microscopy, thus enabling more precise control over protein activity. Therefore, scMagnet activation using two-photon excitation was investigated.

[0207] Because the two-photon absorption cross-section of the flavin cofactor is small, the activation efficiency of two-photon excitation based on the flavin-binding photoswitch containing the Magnet system is very low (Kinjo et al., Nat. Methods 16: 1029-1036 (2019)). To date, there are no examples of effective control of optogenetic rebinding of split proteins using two-photon excitation. Therefore, we consider using the activation induced by two-photon excitation based on the fluorescence resonance energy transfer (FRET) principle (Kinjo et al., Nat. Methods 16: 1029-1036 (2019); Kinjo et al., ACS Chem. Biol. 15: 2848-2853 (2020)) in the scMagnet system. The scMagnet system can effectively perform optogenetic control by connecting pMag and nMag with a flexible connector (see [reference]). Figure 2 c Figure 7 (e.g., a and b). Therefore, an attempt was made to insert the fluorescent protein mTagBFP2 into the linker region connecting pMag and nMag to enable it to function as a FRET donor. Figure 25 ).

[0208] The distance between the two chromophores has a significant impact on FRET efficiency; therefore, a shorter distance between mTagBFP2 and the Magnet system (pMag and nMag) is preferred. Based on the structural models of mTagBFP2 (aa 1-237) and the Magnet system, it was determined that the N-terminal structural domain (aa 1-6) and C-terminal structural domain (aa 229-237) of mTagBFP2 could be missing. Therefore, a two-photon excitation type Vav2 (2PscPA-Vav) was fabricated by inserting ΔmTagBFP2 (aa 7-228) between pMag and nMag into a scMagnet (2PscMagnet). Figure 25 (b and c).

[0209] First, to demonstrate the photoresponsiveness of 2PscPA-Vav, cell stretching was detected using single-photon excitation with 488nm blue light. NIH3T3 cells expressing 2PscPA-Vav exhibited high efficiency in photoinduced stretching of cellular regions. Figure 25 d, Figure 26 ).

[0210] Next, the photoresponsiveness of 2PscPA-Vav was investigated using two-photon excitation microscopy. When two-photon excited with 808 nm near-infrared light, NIH 3T3 cells expressing 2PscPA-Vav exhibited significant stretching of cellular regions compared to the condition maintained in the dark. Figure 25 e and f, Figure 26 The stretching efficiency of cell regions obtained using two-photon excitation was comparable to that obtained using single-photon excitation. On the other hand, even two-photon excitation (15 minutes) of NIH3T3 cells expressing scPA-Vav did not induce stretching of cell regions. Figure 25 g, Figure 27 ).

[0211] The results above show that 2PscMagnet can be activated by two-photon excitation, while scMagnet cannot be activated by two-photon excitation.

[0212] Industrial availability

[0213] According to the present invention, a light-switching protein is provided that allows for precise control of binding and dissociation via light irradiation (ON / OFF). The light-switching protein of the present invention can effectively control the binding and dissociation of various intracellular proteins both in vitro and in vivo. Therefore, it is expected to be used in life science research and in the medical field for the treatment of diseases, etc.

Claims

1. A protein which is a protein in which a C terminus of an (a) protein and an N terminus of an (b) protein are directly or indirectly connected, the (a) protein comprising an amino acid sequence having 80% or more sequence homology with an amino acid sequence obtained by deleting X consecutive amino acid residues from the C terminal side of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 36, and X being an integer of 0 to 6, the (b) protein comprising an amino acid sequence having 80% or more sequence homology with an amino acid sequence obtained by deleting Y consecutive amino acid residues from the N terminal side of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 36, and Y being an integer of 0 to 55, X of the (a) protein being 0 when Y of the (b) protein is an integer of 1 to 55, and Y of the (b) protein being 0 when X of the (a) protein is an integer of 1 to 6. In the amino acid sequence of the (a) protein, when the amino acids corresponding to positions 52 and 55 in the amino acid sequence represented by SEQ ID NO: 1 or the amino acids corresponding to positions 16 and 19 in the amino acid sequence represented by SEQ ID NO: 36 are substituted with arginine, in the amino acid sequence of the (b) protein, the amino acid corresponding to position 52 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with aspartic acid and the amino acid corresponding to position 55 is substituted with glycine, or the amino acid corresponding to position 16 in the amino acid sequence represented by SEQ ID NO: 36 is substituted with aspartic acid and the amino acid corresponding to position 19 is substituted with glycine. The C terminus of the (a) protein and the N terminus of the (b) protein are connected via a polypeptide. wherein The polypeptide is a fluorescent protein or a bioluminescent protein.

2. The protein of claim 1, wherein, The fluorescent protein is any one of BFP, TagBFP, mTagBFP2, mBlueberry2, mCerulean, mTurquoise, ECFP, TagCFP, Rosmarius, meleCFP, mTFPl, KCY, meffCFP, GFP, YFP, Venus, mCherry, and iRFP.

3. The protein of claim 1, wherein, The bioluminescent protein is any one of NanoLu, Gaussia luciferase, Renilla luciferase, and Firefly luciferase.

4. The protein of claim 3, wherein, 7. A nucleic acid encoding the protein according to any one of claims 1 to 6.

5. The protein of claim 4, wherein, In the protein according to any one of claims 1 to 6, a procedure of binding the (a) protein to one of the two proteins, a procedure of binding the (b) protein to the other of the two proteins, and a procedure of irradiating light to the protein according to any one of claims 1 to 6.

6. The protein of claim 4, wherein, The two proteins are interacting proteins.

8. A method for producing a protein according to any one of claims 1 to 6, comprising the steps of: a) introducing a nucleic acid encoding the (a) protein into a host cell, and expressing the (a) protein; b) introducing a nucleic acid encoding the (b) protein into the host cell, and expressing the (b) protein; and c) introducing a nucleic acid encoding a polypeptide into the host cell, and expressing the polypeptide.

8. A method of controlling the distance of two proteins comprising:

9. A method for producing a protein according to any one of claims 1 to 6, comprising the steps of: a) introducing a nucleic acid encoding the (a) protein into a host cell, and expressing the (a) protein; b) introducing a nucleic acid encoding the (b) protein into the host cell, and expressing the (b) protein; and c) introducing a nucleic acid encoding a polypeptide into the host cell, and expressing the polypeptide.

9. The method of claim 8, wherein, 10. A method for producing a protein according to any one of claims 1 to 6, comprising the steps of: a) introducing a nucleic acid encoding the (a) protein into a host cell, and expressing the (a) protein; b) introducing a nucleic acid encoding the (b) protein into the host cell, and expressing the (b) protein; c) introducing a nucleic acid encoding a polypeptide into the host cell, and expressing the polypeptide; and d) introducing a nucleic acid encoding a polypeptide into the host cell, and expressing the polypeptide.

11. A method for producing a protein according to any one of claims 1 to 6, comprising the steps of: a) introducing a nucleic acid encoding the (a) protein into a host cell, and expressing the (a) protein; b) introducing a nucleic acid encoding the (b) protein into the host cell, and expressing the (b) protein; c) introducing a nucleic acid encoding a polypeptide into the host cell, and expressing the polypeptide; d) introducing a nucleic acid encoding a polypeptide into the host cell, and expressing the polypeptide; and e) introducing a nucleic acid encoding a polypeptide into the host cell, and expressing the polypeptide.

12. A method for producing a protein according to any one of claims 1 to 6, comprising the steps of: a) introducing a nucleic acid encoding the (a) protein into a host cell, and expressing the (a) protein; b) introducing a nucleic acid encoding the (b) protein into the host cell, and expressing the (b) protein; c) introducing a nucleic acid encoding a polypeptide into the host cell, and expressing the polypeptide; d) introducing a nucleic acid encoding a polypeptide into the host cell, and expressing the polypeptide; e) introducing a nucleic acid encoding a polypeptide into the host cell, and expressing the polypeptide; and f) introducing a nucleic acid encoding a polypeptide into the host cell, and expressing the polypeptide.

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