CN110468153B

CN110468153B - Genome transcription device regulated by far-red light and based on CRISPR/Cas9 system, construction method and application

Info

Publication number: CN110468153B
Application number: CN201810448630.3A
Authority: CN
Inventors: 叶海峰; 周阳; 邵佳伟; 王美艳; 余贵玲
Original assignee: East China Normal University
Current assignee: East China Normal University
Priority date: 2018-05-11
Filing date: 2018-05-11
Publication date: 2022-12-06
Anticipated expiration: 2038-05-11
Also published as: CN110468153A

Abstract

The invention discloses a genome transcription device regulated by far-red light and based on a CRISPR/Cas9 system, which comprises a far-red light sensing element, a far-red light effect element and a genome positioning transcription element. The genome transcription device based on the CRISPR/Cas9 system and regulated by far-red light can efficiently regulate the transcription expression of endogenous and exogenous genes (single or multiple) of mammals. The system can accurately regulate and control the expression of endogenous and exogenous genes of mammalian cells through far-red light, has the characteristics of no toxicity, high efficiency, insulativity and space-time specificity induced gene expression, and has huge potential application value in the research of accurately regulating and controlling cell behaviors in space-time in the fields of mammalian gene engineering and regenerative medicine.

Description

Genome transcription device regulated by far-red light and based on CRISPR/Cas9 system, construction method and application

Technical Field

The invention relates to the multidisciplinary crossing field of synthetic biology, optogenetics and the like, in particular to a genome transcription device regulated by far-red light and based on a CRISPR/Cas9 system, a construction method, high-efficiency induction of expression of endogenous and exogenous genes of mammalian cells, and application of the genome transcription device in mammalian gene engineering and regenerative medicine.

Background

In the field of synthetic biology, molecular switches for artificially and precisely regulating gene expression have become an indispensable means in mammalian genetic engineering. There are many systems for artificially regulating the expression of an inducible gene, and these regulation systems mainly induce the expression of the inducible gene by a chemical inducer or a physical method, but there are few systems for regulating the expression of an endogenous gene by light induction.

Light is an ideal inducer of gene expression. It is ubiquitous in nature, readily available, spatio-temporal specific, and non-toxic. Therefore, the light is used as an inducer to regulate the expression of endogenous genes and has great application value in the research of genetic engineering.

At present, a CRISPR-based system has been developed to be a powerful gene editing tool, and has a wide application prospect in the biomedical field. These systems, consisting of Cas9 nucleases and single stranded RNAs (sgrnas), enable relatively easy regulation of gene expression, including induction of gene expression, gene silencing, gene defect repair, etc., for a specific gene. Up to now, the transcription system for photoactivating endogenous genes based on the regulation of dCas9 has been deficient. These optically-activated transcription systems based on CRISPR/Cas9 basically use blue light or ultraviolet light as an inducer, but they have certain limitations, such as certain toxicity, low transdermal efficiency, and difficulty in regulating and controlling target genes through skin or abdominal cavity, which greatly limits the deep development and clinical application of the existing optically-controlled CRISPR/Cas9 systems. To overcome these limitations, it is highly desirable to invent a fast-response, convenient, efficient, safe and low-toxicity genome transcription apparatus to effectively transcriptionally activate endogenous gene expression for transformation research and clinical applications.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a genome transcription device regulated by far-red light and based on a CRISPR/Cas9 system, which is used for carrying out induced expression on endogenous genes of mammals.

The invention provides a genome transcription device regulated by far-red light and based on a CRISPR/Cas9 system for the first time. In the invention, the photon energy of far-red light is much lower than that of blue light, and the toxic and side effects on cells are far less than that of blue light. The penetrability of far-red light is far greater than that of blue light, and the far-red light can permeate 7-8 cm of skin and muscle tissues, so that target cells transplanted in an animal body can be controlled to express target genes without traces, and even specific tissues and organs in the animal body can be controlled to express the target genes. In addition, the far-red light control system can be activated by far-red light without adding any photosensitive pigment additionally.

The CRISPR/Cas9 system refers to the condition that a nuclease Cas9 protein can be combined with a tracrRNA (trans-activating RNA) and a compound of complementary pairing thereof, and double-stranded DNA (deoxyribonucleic acid) is cut at a corresponding sequence target site. The dCas9 protein is a mutant of Cas9 protein, has no cleavage activity, but can still accurately target genomic DNA under the mediation of sgRNA.

The invention also provides a control system for regulating the endogenous gene expression loop by far-red light. The invention optimizes far-red light effect elements in the system, achieves the optimal induction effect, makes the system more sensitive to far-red light, and is more beneficial to deep development and clinical application of a future light control system. The far-red light regulation gene expression loop control system can induce the expression of single or multiple endogenous genes, provides a new tool for genetic engineering, has great potential application value, and can be widely popularized in clinical application.

The nucleotide sequences or amino acid sequences can be prepared by adopting an artificial synthesis method.

The invention provides a genome transcription device based on a CRISPR/Cas9 system, which is artificially designed and synthesized and is regulated by far-red light.

The invention also provides a far-red light regulated eukaryotic expression vector and an engineered cell of the genome transcription device based on the CRISPR/Cas9 system.

The invention also provides a kit for each component of the genome transcription device based on the CRISPR/Cas9 system and regulated by the far-red light.

The invention can quickly induce the expression of endogenous genes, can regulate and control the expression quantity of the genes, and has the characteristics of high induced expression multiple, high space-time specificity, strong tissue penetrating power, no toxic or side effect and the like.

The invention provides a far-red light regulated genome transcription device based on a CRISPR/Cas9 system, which comprises: far-red light sensing elements, far-red light effect elements, and genome-specific transcription elements.

In the invention, the far-red light sensing element comprises bacterial photosensitive diguanylate cyclase BphS and degrading enzyme YhjH of c-di-GMP. The amino acid sequence of the BphS is shown in SEQ ID NO.23, and the coding gene sequence of the degrading enzyme YhjH of the c-di-GMP is Genebank accession number: ANK04038.

In the present invention, the far-red light response element comprises a fusion protein P65-VP64-BldD responding to c-di-GMP, and a promoter P for promoting expression of a transcriptional activator _FRL And Transcriptional Activators (FGTAs) that transcriptionally activate downstream genes.

Wherein the sequence of the fusion protein p65-VP64-BldD is shown in SEQ ID NO. 24.

Wherein, the promoter P _FRL A DNA sequence recognized and combined by the BldD protein and a weak promoter for promoting gene expression;

the DNA sequence combined with the BldD protein is a DNA sequence specifically recognized and combined by the polypeptides of the DNA binding domain and the c-di-GMP binding domain, is a partial sequence of a whisG promoter region, and has a nucleotide sequence shown as SEQ ID NO.1 (a nucleotide sequence of a BldD binding site (whisG)), and different copy numbers of whisG;

the weak promoter for starting gene expression comprises TATAbox, a nucleotide sequence is shown as SEQ ID NO.2, a cytomegalovirus hCMV minimum promoter, a nucleotide sequence is shown as SEQ ID NO.3, and a mutant hCMV min3G thereof, and the nucleotide sequence is shown as SEQ ID NO. 4.

The invention optimizes the DNA sequence recognized and combined by BldD in an effector and the weak promoter for promoting gene expression, and the DNA sequence recognized and combined by BldD and the weak promoter for promoting gene expression can be selected from P with the nucleotide sequence shown as SEQ ID NO.5 _FRL1a: pA-3*whiG-P _hCMVmin P shown as SEQ ID NO.6 _FRL2a: 3*whiG-P _hCMVmin P shown as SEQ ID NO.7 _FRL3a: 1*whiG-P _hCMVmin P shown as SEQ ID NO.8 _FRL1b: 2*whiG-P _hCMVmin3G P shown as SEQ ID NO.9 _FRL2b: 3*whiG-P _hCMVmin3G 。

Wherein the protein encoded by the far-red light-induced transcription activator (FGTAs)Are transcriptional activators capable of activating endogenous genes, including various combinations of VP64, p65, MS2, VPR, and HSF 1. Wherein, VP64 is a transcription activation domain of herpes simplex virus particle protein VP16 and 4 copies of the transcription activation domain, and the amino acid sequence is shown in SEQ ID NO. 11. Wherein, the p65 is NF-KB p65 subunit transcription activation structural domain, and the amino acid sequence is shown as SEQ ID NO. 12. Wherein, the MS2 is coat protein, and the amino acid sequence of the coat protein is shown in SEQ ID NO. 13. Wherein VPR is a VP64-p65-Rta fusion protein, and the amino acid sequence of the VPR is shown as SEQ ID NO. 14. Wherein, the HSF1 is a transcription activation domain of a heat shock transcription factor HSF1, and the amino acid sequence of the transcription activation domain is shown as SEQ ID NO. 15. The different combined amino acid sequences can be selected from the sequence SEQ ID NO.16-19, and comprise FGTA1: MS2-Linker-NLS-VP64, FGTA2: MS2-Linker-NLS-VPR, FGTA3: MS2-Linker-NLS-p65-HSF1-VP64, FGTA4: MS2-Linker-NLS-p65-HSF1 and the like. Wherein, when the transcription activator induced by far-red light is FGTA4: MS2-Linker-NLS-P65-HSF1, the promoter is P _FRL1b The induction activation efficiency is highest.

In the present invention, the genome-localized transcription element includes dCas9 protein and single-stranded RNA (sgRNA) having a guide targeting effect.

Wherein the dCas9 is a mutant (D10A, H840A) of the Cas9 protein, and the amino acid sequence of the mutant is shown as SEQ ID NO. 10; the single-stranded RNA includes a gRNA capable of targeting a gene promoter of interest.

The action mechanism of the genome transcription device based on the CRISPR/Cas9 system and regulated by far-red light is that when c-di-GMP is generated under the condition of far-red light irradiation, the c-di-GMP is combined with BldD to form a dimer, a downstream pathway is activated to express a transcription activator, and then the transcription activator is combined with an MS2 stem-loop structure to activate the transcription expression of genes. When the illumination is stopped and the c-di-GMP cannot be generated, the synthesized c-di-GMP is degraded, and the BldD cannot form a dimer, so that the expression of a transcription activator cannot be started, and the target gene cannot be transcribed.

Three components of the far-red light regulated and controlled genome transcription device based on the CRISPR/Cas9 system can be constructed in a eukaryotic expression vector through a genetic engineering technology, so that the transcription expression of a regulated and controlled target gene is realized. The genome transcription device regulated by far-red light and based on the CRISPR/Cas9 system can specifically induce the expression of endogenous genes in eukaryotic host cells in time and space by using the far-red light which hardly damages cells or organisms, and the host cells can be any types of mammalian cells, such as hMSC-TERT, hana3A, HEK-293A, HEK-293T and the like.

Wherein the illumination intensity of the far-red light is 0-5mW/cm ² (ii) a The irradiation time is 0-72h; the irradiation method includes pulsed irradiation, continuous irradiation, or direct irradiation to spatially control the gene expression levels of cells at different positions. The far-red light source is controlled to generate different illumination time, so that the induction of endogenous genes in different degrees is realized. The far-red light source can generate 600-900nm far-red light, and can be 600-900nm LED, infrared therapeutic equipment, laser lamp, etc.

The invention optimizes far-red light effect elements, including a promoter P for promoting expression of a transcriptional activator _FRL And Transcriptional Activators (FGTAs) that transcriptionally activate downstream genes. (1) The invention optimizes the promoter P _FRL Wherein, the promoter P _FRL Comprising a DNA sequence to which BldD dimerizes under the action of c-di-GMP recognizes and binds and a weak promoter sequence which promotes gene expression. The weak promoter for promoting gene expression can be any weak promoter, such as TATA box with a nucleotide sequence shown as a sequence SEQ ID NO.2 and cytomegalovirus minimal promoter hCMV with a nucleotide sequence shown as a sequence SEQ ID NO.3 _min And mutant hCMVmin3G, and the like, and the nucleotide sequence is shown in SEQ ID NO. 4. The DNA sequence and the promoter identified and combined by the BldD can be selected from P with the nucleotide sequence shown as SEQ ID NO.5 _FRL1a: pA-3*whiG-P _hCMVmin P shown as SEQ ID NO.6 _FRL2a: 3*whiG-P _hCMVmin P shown as SEQ ID NO.7 _FRL3a: 1*whiG-P _hCMVmin P shown as SEQ ID NO.8 _FRL1b: 2*whiG-P _hCMVmin3G P shown as SEQ ID NO.9 _FRL2b: 3*whiG-P _hCMVmin3G The system can be made more fold-induced.(2) The invention optimizes the transcription activator which can be selected from FGTA1: MS2-Linker-NLS-VP64, FGTA2: MS2-Linker-NLS-VPR, FGTA3: MS2-Linker-NLS-p65-HSF1-VP64, FGTA4: MS2-Linker-NLS-p65-HSF1 and the like, and the amino acid sequence is shown in SEQ ID NO.16, 17, 18 and 19.

The invention also provides a construction method of the far-red light regulated and controlled genome transcription device based on the CRISPR/Cas9 system, which comprises the following steps:

(1) Construction of far-red photoreceptors

A complex of a polypeptide as a DNA binding domain and a c-di-GMP binding domain, a polypeptide as a nuclear localization signal NLS, a polypeptide as a linking domain, and a polypeptide as a transcription regulatory domain is constructed as a far-red light sensing element of the system.

Wherein, the polypeptide as the DNA binding domain and the c-di-GMP binding domain is a protein which can be combined with a specific DNA sequence after being combined with the c-di-GMP, and comprises a BldD protein, and the amino acid sequence of the BldD protein is shown in SEQ ID NO. 20;

wherein, the polypeptide as the nuclear localization signal NLS can be in various forms of 1-3 copies, and the amino acid sequence of the polypeptide is shown as SEQ ID NO. 21;

wherein, the polypeptide as the connecting functional domain can be in various forms with the length of 0-30 amino acids, and the amino acid sequence is shown as SEQ ID NO. 22;

wherein the polypeptide as a transcription regulatory domain is a domain protein having a transcription activation function.

Wherein the polypeptide as a transcriptional regulatory domain is placed N-terminal or C-terminal to the DNA binding domain and the polypeptide BldD of the C-di-GMP binding domain.

(2) And constructing a far-red light effect element.

Wherein the far-red light response element comprises promoter P _FRL And Transcriptional Activators (FGTAs), denoted P _FRL -FGTAs. The promoter P _FRL May also be a BldD protein-binding DNA sequence and a weak promoter, wherein the processor is a DNA sequence specifically recognized and bound by the polypeptides of the DNA-binding domain and the c-di-GMP-binding domain, and is a DNA sequence specifically recognized and bound by the BldD protein-binding DNA sequence and the weak promoterPartial sequence of whisG promoter region, nucleotide sequence is shown in SEQ ID NO.1, and its different copy forms; the weak promoters that initiate gene expression include all weak promoters including TATAbox, cytomegalovirus CMV minimal promoter and its mutant CMVmin 3G.

(3) A genome-localized transcription element is constructed.

Wherein the genome-localized transcription element comprises dCas9 protein and single-stranded RNA (sgRNA) with a targeting guiding effect;

wherein the dCas9 is a mutant D10A and H840A of the Cas9 protein, and the amino acid sequence of the mutant is shown as a sequence SEQ ID NO. 10.

The single-stranded RNA (sgRNA) having a targeting guidance function is an arbitrary RNA sequence that can bind to a promoter of a target gene and bind to dCas9 protein.

The invention also provides a kit which contains the far-red light regulated and controlled genome transcription device based on the CRISPR/Cas9 system. The invention also provides a kit which contains the eukaryotic expression vector of the genome transcription device based on the CRISPR/Cas9 system regulated by the far-red light and/or a host cell transfected with the eukaryotic expression vector and/or an engineered cell transplantation vector and a corresponding instruction.

In the invention, the kit comprises a plasmid kit for regulating and controlling each component of the far-red light regulation and control gene expression loop control system, a mammalian cell kit containing the far-red light regulation and control gene expression loop control system, and a corresponding instruction manual.

The invention also provides a method for preparing the eukaryotic expression vector, the engineered cell or the engineered cell transplantation vector of the genome transcription device based on the CRISPR/Cas9 system and regulated by the far-red light.

The eukaryotic expression vector comprises a mammalian cell expression vector containing the far-red light-regulated CRISPR/Cas9 system-based genome transcription device. The expression vector can be a vector containing a far-red light sensing element coding gene alone or a vector containing a far-red light effect element coding gene alone or a vector containing a genome mapping gene aloneA vector of a gene encoding a transcription element comprising a far-red light-responsive promoter (P) _FRL ) And Transcriptional Activator (FGTA). Or the expression vector comprises two or three of a vector of the far-red light sensing element coding gene, a vector of the far-red light effect element coding gene and a vector of the genome positioning transcription element coding gene. The construction of all the aforementioned mammalian cell expression vectors is detailed in Table 1.

The invention also provides application of the eukaryotic expression vector of the genome transcription device based on the CRISPR/Cas9 system and regulated by the far-red light in inducing the expression of endogenous genes of mammals.

The invention has the beneficial effects that: the genome transcription device regulated by the far-red light and based on the CRISPR/Cas9 system can quickly induce the expression of endogenous genes through the far-red light, accurately regulate and control the gene expression, and has the characteristics of high induced gene expression multiple, high space-time specificity, strong tissue penetration, no toxic or side effect and the like.

Drawings

FIG. 1 is a schematic diagram of a genome transcription device based on CRISPR/Cas9 system for far-red light regulation;

fig. 2 is a graph of the results of the activation efficiency of different transcription activators for a far-red light regulated CRISPR/Cas9 system based genome transcription device;

FIG. 3 is a graph of the results of CRISPR/Cas9 system-based genome transcription device-induced activation efficiency for far-red light regulation by different promoters;

FIG. 4 is a graph of the results of CRISPR/Cas9 system-based genome transcription device-induced activation efficiency for far-red light regulation at different illumination intensities;

fig. 5 is a graph of the results of the activation efficiency of a CRISPR/Cas9 system-based genome transcription device with different illumination times for far-red light regulation;

fig. 6 is a graph of the results of the activation efficiency of a CRISPR/Cas9 system-based genome transcription apparatus for far-red light regulation in different cell lines;

fig. 7 is a verification of the reversible regulation of a genome transcription apparatus based on CRISPR/Cas9 system with far-red light regulation;

FIG. 8 is a spatial specificity validation of a CRISPR/Cas9 system-based genome transcription apparatus for far-red light regulation;

fig. 9 is a graph of the results of the efficiency of the endogenous gene activated induced by the CRISPR/Cas9 system-based genome transcription apparatus for far-red light regulation by different grnas;

fig. 10 is a graph of the results of the efficiency of endogenous gene activation induced by a CRISPR/Cas9 system-based genome transcription apparatus with different illumination intensities for far-red light regulation;

fig. 11 is a graph of the results of the efficiency of activating endogenous genes of a CRISPR/Cas9 system-based genome transcription apparatus regulated by far-red light at different illumination times;

fig. 12 is a graph of the results of the efficiency of activating endogenous genes in different cell lines for a CRISPR/Cas9 system-based genome transcription apparatus regulated by far-red light;

FIG. 13 is a verification of the reversible regulation of the far-red light regulated CRISPR/Cas9 system-based genome transcription device activating endogenous genes;

fig. 14 is a graph of the results of the efficiency of a genome transcription apparatus based on CRISPR/Cas9 system under far-red light regulation to simultaneously activate multiple endogenous genes.

Detailed Description

The present invention will be described in further detail with reference to the following specific examples and the accompanying drawings. These examples are intended to illustrate the invention and do not limit the scope of the invention in any way. The procedures, conditions, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited. The reagents, instruments, etc. used in the following examples, as well as the experimental methods not specified for specific conditions, were conducted according to the conditions conventionally or proposed by commercial suppliers.

Materials and methods

Molecular cloning

The molecular cloning technology is used for constructing all expression plasmids of the invention, and the steps are common knowledge in the field.

All primers used for PCR were synthesized by Kingzhi Biotech, inc. The expression plasmids constructed in the examples of the present invention were subjected to sequence determination, which was accomplished by Jinzhi Biotechnology, inc. Phanta Max Super-Fidelity DNA polymerase used in the examples of the present invention was purchased from Biotech, inc. of Nanjing Novowed Toxa. The endonuclease and the T4DNA ligase were purchased from TaKaRa. Homologous recombinases were purchased from Heyu Biotechnology (Shanghai) Ltd. Phanta Max Super-Fidelity DNA polymerase was purchased with the corresponding polymerase buffer and dNTPs. The endonuclease, T4DNA ligase and homologous recombinase were purchased with the corresponding buffer. Yeast Extract (Yeast Extract), tryptone (Trypton), agar powder, and ampicillin (Amp) were obtained from Shanghai Biotech, inc. DNA Marker DL5000, DNAMarker DL2000 (baobaozhen bio engineering ltd); nucleic acid dye EB (guangdong national ao biotechnology); a plasmid small extraction kit (Tiangen Biochemical technology (Beijing) Co., ltd.); the DNA gel recovery kit and the PCR product purification kit are purchased from Shikoku Biotechnology GmbH; the other reagents such as absolute ethyl alcohol, naCl and the like mentioned in the examples are all domestic analytical pure products. The method comprises the following steps of (1) glue recovery and purification recovery of DNA fragments, wherein the steps are carried out according to the operation instructions of a DNA glue recovery kit and a PCR product purification kit (Kangji Biotech Co., ltd.); plasmid extraction procedure was performed according to the plasmid Mini-extraction kit (Tiangen Biochemical technology (Beijing) Ltd.).

Cell culture and transfection

The following cell line and PEI transfection are used as examples in the examples of the present invention to illustrate the working of the far-red light-regulated genome transcription apparatus based on CRISPR/Cas9 system in cells, but not to limit the scope of the present invention.

10cm cell culture dishes, cell culture plates (24 wells), 15mL and 50mL centrifuge tubes for cell culture were purchased from Thermo Fisher Scientific, USA (Labserv); modified Eagle Medium, fetal bovine serum, penicillin and streptomycin solutions used were purchased from Gibico, USA; PEI used for transfection was purchased from Polysciences; cell culture chambers were purchased from Thermo Fisher Scientific, usa; the other consumables are common domestic consumables.

Cell culture: the cells involved in the patent include human embryonic kidney cells (HEK-293, ATCC, CRL-11268) stably incorporating a copy of the E1 gene (ThermoFisher, R70507), telomeric human mesenchymal stem cells (hmsct-tert 26) and HEK-293 derived Hana3A cells, all cultured in modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin and streptomycin solutions; the cells were cultured in an incubator containing 5% carbon dioxide at 37 ℃.

Transfection: all cell lines were transfected using the procedure for optimized PEI (Wieland M, methods 56 (3): 351). Briefly, the cells were inoculated in a 10-cm cell culture dish having a culture system of 10mL, and the inoculation is carried out at 10X ⁴ After culturing for 18h, the cells were mixed with PEI and dissolved in the culture medium at an optimum ratio of DNA to PEI according to a mass ratio of 3 (PEI: DNA) for standing for 6h. (polyethyleneimine, molecular weight 40,000, stock solution 1mg/mL in ddH ₂ O; polysciences; cat No. 24765) cell number was counted using a counter II automated cell counter.

Detection of reporter Gene secreted alkaline phosphatase (SEAP)

Homoarginine, magnesium chloride, diethanolamine and HCl for preparing a detection reporter gene reaction buffer solution are purchased from Biotechnology engineering (Shanghai) GmbH; chromogenic substrate (p-nitrophenol phosphate) was purchased from Shanghai Crystal pure science and technology, inc. (Aladdin).

(1) Reagent preparation:

2 × buffer solution:

20mM homoarginine notes: the function of the inhibitor is to inhibit the activity of endogenous alkaline phosphatase

1mM magnesium chloride

2% diethanolamine

Adjusting the pH to 9.8 with HCl

Substrate solution:

120mM chromogenic substrate (p-nitrophenol phosphate)

2 × assay buffer

(2) The experimental steps are as follows:

1. the cell culture supernatant was aspirated into a centrifuge tube at 200. Mu.L (note: generally more than 150. Mu.L, since a portion of the volume was lost by subsequent heating).

Water bath at 2.65 deg.c for 30min (note: heating mainly inactivates endogenous alkaline phosphatase, while SEAP is high temperature resistant and does not inactivate at this temperature).

3. Pipette 80. Mu.L (diluted by itself depending on the experimental conditions) into a 96-well plate, and add rapidly 100. Mu.L of 2xbuffer and 20. Mu.L of the substrate solution, which have been preheated beforehand.

4. The enzyme-linked immunosorbent assay is carried out 10 times at 405nm, and each time interval is 1min (conditions can be set according to the experiment situation).

(3) Calculation of enzyme Activity

The enzyme activity of alkaline phosphatase (SEAP) is defined as the reaction between the substrate disodium p-nitrophenylphosphate (PNPP-Na) and the substrate disodium p-nitrophenylphosphate (PNPP-Na) at 37 ℃ and pH 9.8 within 1min ₂ ) The reaction produced 1mol/L of p-nitrophenol, defined as 1 activity unit (1U). The p-nitrophenol has bright yellow, and at the wavelength of 405nm, the p-nitrophenol with different concentrations corresponds to different light absorption values. The calculation method comprises the following steps: the slope 256.8 of the curve formed by the OD values measured at different time points in the reaction process of the sample and the substrate is the enzyme activity in U/L.

Gene activation in far-red light controlled mammalian cells

Endogenous gene activation experiments. The culture system was 10mL and the culture system was inoculated into a 10cm cell culture dish at 6X 10 ⁴ And culturing the cells for 18h. Cells were transfected with 50. Mu.l of PEI of 3. 24h after transfection, custom 4-by-6 arrays of far-red LEDs (730nm, epistar, taiwan, china) were used at different illumination times (0-6 h), illumination intensities (0-2 mW/cm) ² ) Light irradiation is performed. SEAP values were determined 48h after transfection. HEK-293 cells were co-transfected with pWS46, pGY32, pGY102, pSZ69 (100 ng each) and sgRNAs (50 ng) followed by 6h of light daily ((1.5 mW/cm) ² (ii) a 730 nm)). Total RNA was extracted 48h after the first illumination and real-time fluorescent quantitative PCR was performed.

Spatial specificity of gene activation in far-red light controlled mammalian cells

3X 10 species in a 10cm diameter petri dish ⁶ HEK293 cells, after 18h incubation, transfected with 1500. Mu.l of 3. 18h after transfection, far-red light (730nm, 1.5mW/cm) was used through a designed photomask ² ) The light is irradiated for 6h. After 48h of light illumination, the fluorescence signal was measured with a Clinx imaging apparatus (ChemiScope 4300Pro, clinx, shanghai, china).

Quantitative RT-PCR analysis

Total RNA was extracted using an RNAlso Plus kit (Takara, dalian, china; cat. No. 9108) collection cell following kit procedures. Mu.g of RNA was reverse transcribed into cDNA using the PrimeScript RT reagent Kit with the gDNA Eraser (Takara, dalian, china; cat. No. RR047) according to the procedure. The target gene was assayed by Real-Time PCR Instrument (QuantStudio 3, thermo Fisher Scientific Inc., waltham, MA, USA) using SYBR Premix Ex Taq (Takara, dalian, china; cat. No. RR420) for qPCR analysis. PCR amplification conditions were first pre-denaturation at 95 ℃ for ten minutes, followed by 40 cycles (denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 30 s) and final extension at 72 ℃ for 10min. All samples were referenced to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the results were expressed as relative RNA levels with reference to dark conditions.

Example 1

In the example, SEAP is taken as a reporter gene to verify the gene activation efficiency of different transcription activators of the genome transcription device based on the CRISPR/Cas9 system regulated by far-red light, but the protection scope of the invention is not limited. The method comprises the following specific steps:

in the first step, plasmid construction. The plasmid construction in this example is detailed in Table 1.

Second, cells are seeded. Good HEK-293T cells in growth state were seeded in 2 24-well plates with 6X 10 cells per well after digestion with 0.25% pancreatin ⁴ Cells were cultured and 500. Mu.L of 10% FBS-containing DMEM medium was added.

And thirdly, transfection. The 2 24-well plates were divided into dark and light groups, each group being 4 subgroups. Within 16 to 24h of seeding the cells, pWS46 (P) was added to each group _hCMV -BphS-2A-YhjH-pA),pGY32(P _hCMV -FRTA3-pA),pSZ69(P _hCMV -dCas9-pA),pWS137(P _U6 -gRNA1(P _FACE )-pA)，pWS107(P _FACE SEAP-pA) and the different transcriptional activators FGTA1 (pGY 48) or FGTA2 (pGY 51) or FGTA3 (pGY 54) or FGTA4 (pGY 57), were mixed with PEI transfection reagent and serum-free DMEM in the ratio of 2. Wherein the total preparation volume of each hole is 50 mu L, and the mass ratio of the plasmid to the PEI is 1. 6h after transfection, the cells were cultured by changing into 500. Mu.L of DMEM medium containing 10% FBS.

And fourthly, illuminating. After the liquid is changed for 14-18h, the illumination group is placed at a wavelength of 730nm and the illumination intensity is respectively 1.5mW/cm ² The LED of (1) is illuminated for 6h, and the dark group is always cultured in the dark.

And fifthly, detecting the reporter gene.

The result shows that in the genome transcription device based on the CRISPR/Cas9 system and regulated by far-red light, the activation efficiency is most remarkable when the activator is FGTA 4. Experimental data are detailed in figure 2 of the specification, all presented as n =3 independent replication experiments.

Example 2

In the example, SEAP is used as a reporter gene to verify the gene activation efficiency of different promoters in the genome transcription device based on the CRISPR/Cas9 system regulated by far-red light, but the protection scope of the invention is not limited. The method comprises the following specific steps:

In the second step, cells are seeded. (the procedure is as in example 1)

And thirdly, transfection. The 2 24-well plates were divided into a dark group and an illuminated group, each group divided equally into 5 small groups. Within 16 to 24h of the inoculated cells, pWS46 (100 ng), pGY32, pWS137, pWS107, pSZ69 and the different promoters P were added to each group _FRL1a [pA-(whiG) ₃ -P _hCMVmin ；pGY59]Or P _FRL2a [(whiG) ₃ -P _hCMVmin ；pGY58]Or P _FRL3a [(whiG)-P _hCMVmin ；pGY57]Or P _FRL1b [(whiG) ₂ -P _hCMVmin3G ；pGY102]Or P _FRL2b [(whiG) ₃ -P _hCMVmin3G ；pGY77]The expressed transcriptional activator FGTA4 (MS 2-p65-HSF 1), at the ratio of 4. Wherein the total preparation volume of each hole is 50 mu L, and the mass ratio of the plasmid to the PEI is 1. 6h after transfection, the cells were cultured in 500. Mu.L of DMEM medium containing 10% FBS.

And fourthly, illuminating. (the concrete procedure was the same as in example 1)

And fifthly, detecting the reporter gene.

The result shows that when the promoter is P in the genome transcription device based on the CRISPR/Cas9 system and regulated by far-red light _FRL1b [(whiG) ₂ -P _hCMVmin3G ；pGY102]The activation efficiency is most pronounced. Experimental data are detailed in figure 3 of the specification, and all data are presented as n =3 independent replication experiments.

Example 3

In the example, SEAP is used as a reporter gene to verify the activation efficiency of different illumination intensities of the genome transcription device based on the CRISPR/Cas9 system regulated by far-red light, but the protection scope of the invention is not limited. The method comprises the following specific steps:

In the second step, cells are seeded. Good HEK-293 cells in growth state were seeded in 7 24-well plates 6X 10 per well after digestion with 0.25% pancreatin ⁴ Cells were cultured and 500. Mu.L of 10% FBS-containing DMEM medium was added.

And thirdly, transfection. Divided into 7 subgroups. Within 16 to 24h of seeding the cells, the addition of pWS46, pGY32, pGY102, pSZ69, pWS137 and pWS107 to each group was mixed with PEI transfection reagent and serum-free DMEM at a ratio of 4. Wherein the total preparation volume of each hole is 50 mu L, and the mass ratio of the plasmid to the PEI is 1. 6h after transfection, the cells were cultured by changing into 500. Mu.L of DMEM medium containing 10% FBS.

And fourthly, illuminating. After the liquid is changed for 14-18h, the solution is placed at the wavelength of 730nm under the illumination intensityThe degrees are respectively 0, 0.025, 0.05, 0.75, 1, 1.5 and 2mW/cm ² The LED is used for illuminating for 4 hours, and the cultivation is carried out in the dark after the illumination is finished.

And step five, detecting the reporter gene.

The result shows that in the genome transcription device based on the CRISPR/Cas9 system regulated by far-red light, the illumination intensity is in positive correlation with the activation efficiency and is 1mW/cm ² Above which the activation efficiency slowly approaches saturation. Experimental data are detailed in figure 4 of the specification, all presented as n =3 independent replication experiments.

Example 4

In the example, SEAP is used as a reporter gene to verify the activation efficiency of the genome transcription apparatus based on CRISPR/Cas9 system for regulating and controlling far-red light at different illumination times, but the protection scope of the invention is not limited. The method comprises the following specific steps:

Second, cells are seeded. Good HEK-293 cells in growth state were seeded in 7 24-well plates 6X 10 per well after digestion with 0.25% pancreatin ⁴ Cells were cultured and 500. Mu.L of 10% FBS-containing DMEM medium was added.

And fourthly, illuminating. After the liquid is changed for 14-18h, the solution is placed at the wavelength of 730nm and the illumination intensity of 1.5mW/cm ² Respectively illuminating for 0, 0.25, 0.5, 1, 2, 4 and 6 hours under the LED (the specific connection mode refers to materials and methods), and immediately culturing in the dark after illumination is finished.

And fifthly, detecting the reporter gene.

The result shows that in the genome transcription device version 2.2 based on the CRISPR/Cas9 system regulated by far-red light, the illumination time is in positive correlation with the activation efficiency, the activation efficiency slowly approaches to saturation after 4 hours, and the maximum activation efficiency is achieved after 6 hours of illumination. Experimental data are detailed in figure 5 of the specification, and all data are presented as n =3 independent replication experiments.

Example 5

The example uses SEAP as a reporter gene to verify the activation efficiency of the genome transcription apparatus based on CRISPR/Cas9 system regulated by far-red light in different cell lines, but does not limit the protection scope of the present invention. The method comprises the following specific steps:

In the second step, cells are seeded. The well grown HeLa cells, HEK-293A cells, hana3A cells, hmsc-TERT cells, HEK-293 cells were digested with 0.25% trypsin and seeded in 2 24-well plates, 6X 10/well ⁴ Cells were plated and 500. Mu.L of DMEM medium containing 10% FBS was added.

And thirdly, transfection. The 2 24-well plates were divided into dark and light groups, each of which was divided into 5 subsets with different cell lines. Within 16 to 24h of seeding the cells, pWS46, pGY32, pGY102, pSZ69, pWS137 and pWS107 were added to each group, mixed with PEI transfection reagent and serum-free DMEM at a ratio of 4. Wherein the total preparation volume of each hole is 50 mu L, and the mass ratio of the plasmid to the PEI is 1. 6h after transfection, the cells were cultured in 500. Mu.L of DMEM medium containing 10% FBS.

And fourthly, illuminating. After the liquid is changed for 14-18h, the illumination group is placed at the wavelength of 730nm and the illumination intensity is 1.5mW/cm respectively ² The LED of (1) was illuminated for 6h, while the dark group was kept in the dark for cultivation.

And fifthly, detecting the reporter gene.

The results show that the far-red light regulated genome transcription device version 2.2 based on the CRISPR/Cas9 system has the best activation efficiency in HEK-293 cells. Experimental data are detailed in figure 6 of the specification, all data are presented as n =3 independent replication experiments.

Example 6

In the example, SEAP is used as a reporter gene to verify the reversibility of the CRISPR/Cas9 system-based genome transcription device activating gene regulated by far-red light, but the scope of the invention is not limited. The method comprises the following specific steps:

And thirdly, transfection. The 2 24-well plates were divided into two groups. Within 16 to 24h of seeding the cells, pWS46, pGY32, pGY102, pSZ69, pWS137 and pWS107 were added to each group in a ratio of 4. Wherein the total preparation volume of each hole is 50 mu L, and the mass ratio of the plasmid to the PEI is 1. 6h after transfection, the cells were cultured in 500. Mu.L of DMEM medium containing 10% FBS.

And fourthly, illuminating. After the liquid is changed for 14-18h, the first group is placed at the wavelength of 730nm and the illumination intensity of 1.5mW/cm ² The culture medium is irradiated under the LED (the specific connection mode refers to the materials and the method) for 20min, the culture medium is immediately placed in a dark place for culture after the illumination is finished, the culture medium is continuously irradiated for 20min on the third day, the culture medium is firstly placed in the dark place for culture on the second group, and the culture medium is irradiated for 20min on the second day and then placed in the dark place for culture.

And fifthly, detecting the reporter gene every 6h and replacing the culture medium every 24 h.

The result shows that the genome transcription device based on the CRISPR/Cas9 system and regulated by far-red light has good reversible regulation. Experimental data are detailed in figure 7 of the specification, all presented as n =3 independent replication experiments.

Example 7

In the example, GFP is used as a reporter gene to verify the spatial specificity of the gene expression induced by the far-red light-regulated CRISPR/Cas9 system-based genome transcription device, as shown in the left part of the attached figure 8, but the protection scope of the invention is not limited. The method comprises the following specific steps:

In the second step, cells are seeded. Good HEK-293 cells in growth state were seeded in two petri dishes after 0.25% trypsin digestion, and 3.5X 10 cells were seeded, respectively ⁶ Cells, and 15mL of DMEM medium containing 10% FBS was added.

And thirdly, transfection. The dishes were divided into two groups, light and dark, and pWS46, pSTING, pSZ70, pSZ69, pWS137 and pGY47 (P) were added to each group _FACE EGFP-pA) was mixed with PEI transfection reagent and serum-free DMEM at a ratio of 10. The cells were cultured 6 hours after transfection in 15mL of DMEM medium containing 10% FBS.

And fifthly, detecting an experimental result. The cells in the dish were observed under a fluorescent microscope and photographed.

The result shows that the genome transcription device based on the CRISPR/Cas9 system and regulated by far-red light can induce gene expression in a space-specific manner. Experimental data are detailed in figure 8 of the specification, and all data are presented as n =3 independent replication experiments.

Example 8

In the example, ASCL1 is used as a reporter gene to verify the influence of gRNA on activation efficiency in a genome transcription device based on a CRISPR/Cas9 system and regulated by far-red light, but the protection scope of the invention is not limited. The method comprises the following specific steps:

Second, cells are seeded. Good HEK-293 cells in growth state were seeded in 2 24-well plates 6X 10 per well after 0.25% trypsin digestion ⁴ Isolating the cells and adding 500 μ L of DMEM medium containing 10% FBS。

Third, 2 24-well plates were divided into dark and light groups, each group being 4 groups. Within 16 to 24h of the inoculated cells, pWS46, pGY32, pGY102, pSZ69 was added to the first group, and pWS46, pGY32, pGY102, pSZ69, pSZ83 (P) was added to the second group _U6 gRNA1 (ASCL 1) -pA), and pWS46, pGY32, pGY102, pSZ69, pSZ84 (P) were added to the third group _U6 -gRNA2 (ASCL 1) -pA), and in the fourth group pWS46, pGY32, pGY102, pSZ69, pSZ83 (P) was added _U6 -gRNA1(ASCL1)-pA),pSZ84(P _U6 -gRNA2 (ASCL 1) -pA), mixed with PEI transfection reagent and serum-free DMEM, left to stand at room temperature for 15min and added to 24-well culture plates. Wherein the total preparation volume of each hole is 50 mu L, and the mass ratio of the plasmid to the PEI is 1. 6h after transfection, the cells were cultured by changing into 500. Mu.L of DMEM medium containing 10% FBS.

And fourthly, illuminating. After the liquid is changed for 14-18h, the illumination group is placed at a wavelength of 730nm and the illumination intensity is respectively 1.5mW/cm ² The LED of (1) was illuminated for 6h, while the dark group was kept in the dark for cultivation.

And fifthly, detecting the reporter gene. Real-time fluorescent quantitative PCR.

The result shows that two gRNAs have better activation efficiency when added into a genome transcription device version 2.2 based on a CRISPR/Cas9 system controlled by far-red light. Experimental data are detailed in figure 9 of the specification, all data are presented as n =3 independent replication experiments.

Example 9

The ASCL1 is taken as a reporter gene in the example to verify the activation efficiency of the genome transcription device based on the CRISPR/Cas9 system and regulated by far-red light for different illumination intensities, but the protection scope of the invention is not limited. The method comprises the following specific steps:

In the second step, cells are seeded. Good HEK-293 cells in growth state were seeded in 7 24-well plates 6X 10 per well after 0.25% trypsin digestion ⁴ Cells were cultured and 500. Mu.L of 10% FBS-containing DMEM medium was added.

And thirdly, transfection. Divided into 7 subgroups. Within 16 to 24h of seeding cells, pWS46, pGY32, pGY102, pSZ69, pSZ83 and pSZ84 were added to each group in a ratio of 4. Wherein the total preparation volume of each hole is 50 mu L, and the mass ratio of the plasmid to the PEI is 1. 6h after transfection, the cells were cultured in 500. Mu.L of DMEM medium containing 10% FBS.

And fourthly, illuminating. After the liquid is changed for 14-18h, the solution is placed at a wavelength of 730nm under the conditions that the illumination intensity is respectively 0, 0.025, 0.05, 0.75, 1, 1.5 and 2mW/cm ² The LED is used for illuminating for 4 hours, and the cultivation is carried out in a dark place after the illumination is finished.

And fifthly, detecting the reporter gene. And (3) real-time fluorescence quantitative PCR.

The result shows that in the genome transcription device based on the CRISPR/Cas9 system regulated by far-red light, the illumination intensity is in positive correlation with the activation efficiency and is 2mW/cm ² The activation efficiency is most significant. Experimental data are detailed in figure 10 of the specification, and all data are presented as n =3 independent replication experiments.

Example 10

The ASCL1 is taken as a reporter gene in the example, and the activation efficiency of the genome transcription device based on the CRISPR/Cas9 system for regulating and controlling the far-red light is verified by different illumination time, but the protection scope of the invention is not limited. The method comprises the following specific steps:

And fourthly, illuminating. After the liquid is changed for 14-18h, the solution is placed at the wavelength of 730nm and the illumination intensity of 1.5mW/cm ² The LED lights for 0, 0.25, 0.5, 1, 2, 4 and 6 hours, and the cultivation is carried out in the dark immediately after the light is finished.

And fifthly, detecting the reporter gene.

The result shows that the far-red light regulated and controlled genome transcription device based on the CRISPR/Cas9 system has the advantages that the illumination time is in positive correlation with the activation efficiency, and the activation effect is most obvious when the illumination time is 6h. Experimental data are detailed in figure 11 of the specification, all presented as n =3 independent replication experiments.

Example 11

The ASCL1 is taken as a reporter gene in the example to verify the activation efficiency of the genome transcription device based on the CRISPR/Cas9 system for far-red light regulation in different cell lines, but the protection scope of the invention is not limited. The method comprises the following specific steps:

In the second step, cells are seeded. HeLa cells, HEK-293A cells, hana3A cells, hMSC-TERT cells, and HEK-293 cells in good growth state were digested with 0.25% trypsin and seeded in 2 24-well plates of 6X 10 cells per well ⁴ Cells were cultured and 500. Mu.L of 10% FBS-containing DMEM medium was added.

And thirdly, transfection. The 2 24-well plates were divided into dark and light groups, each of which was divided into 5 subsets with different cell lines. Within 16 to 24h of seeding cells, pWS46, pGY32, pGY102, pSZ69, pSZ83 and pSZ84 were added to each group, mixed with PEI transfection reagent and serum-free DMEM at a ratio of 4. Wherein the total preparation volume of each hole is 50 mu L, and the mass ratio of the plasmid to the PEI is 1. 6h after transfection, the cells were cultured in 500. Mu.L of DMEM medium containing 10% FBS.

And fourthly, illuminating. After the liquid is changed for 14-18h, the illumination group is placed at a wavelength of 730nm and the illumination intensity is respectively 1.5mW/cm ² The LED under the lamp is illuminated for 6h, and the dark group is always kept in the darkAnd (4) culturing.

And step five, detecting the reporter gene.

The results show that the genome transcription device based on the CRISPR/Cas9 system and regulated by far-red light has the best activation efficiency in HEK-293 cells. Experimental data are detailed in the specification, fig. 12, and all data are presented as n =3 independent replication experiments.

Example 12

In the example, ASCL1 is used as a reporter gene to verify the reversibility of the far-red light regulated genome transcription device activating gene based on the CRISPR/Cas9 system, but the protection scope of the invention is not limited. The method comprises the following specific steps:

Second, cells are seeded. Good HEK-293 cells in growth state were seeded in 2 24-well plates with 6X 10 cells per well after digestion with 0.25% pancreatin ⁴ Cells were cultured and 500. Mu.L of 10% FBS-containing DMEM medium was added.

And thirdly, transfection. The 2 24-well plates were divided into two groups. Within 16 to 24h of seeding cells, pWS46, pGY32, pGY102, pSZ69, pSZ83 and pSZ84 were added to each group in a ratio of 4. Wherein the total preparation volume of each hole is 50 mu L, and the mass ratio of the plasmid to the PEI is 1. 6h after transfection, the cells were cultured in 500. Mu.L of DMEM medium containing 10% FBS.

And fourthly, illuminating. After the liquid is changed for 14-18h, the first group is placed at the wavelength of 730nm and the illumination intensity of 1.5mW/cm ² The LED is used for illuminating for 20min, the culture is immediately carried out in the dark after the illumination is finished, the illumination is continued for 20min after 48h, the second group is firstly placed in the dark for culture for 24h and then is illuminated for 20min, and then is placed in the dark for culture.

The result shows that the genome transcription device based on the CRISPR/Cas9 system and regulated by far-red light has good reversible regulation. Experimental data are detailed in fig. 13 of the specification, all data are presented as n =3 independent replication experiments.

Example 13

In the example, TTN, RHOXF2, IL1RN and ASCL1 are used as reporter genes to verify the efficiency of activating multiple endogenous genes by the CRISPR/Cas9 system-based genome transcription device regulated by far-red light, but the protection scope of the invention is not limited. The method comprises the following specific steps:

Second, cells are seeded. Good HEK-293 cells in growth state were seeded in 14 24-well plates 6X 10 per well after digestion with 0.25% pancreatin ⁴ Cells were cultured and 500. Mu.L of 10% FBS-containing DMEM medium was added.

And thirdly, transfection. The 14 24-well plates were divided into dark and light groups, each divided into 5 sub-groups. Within 16 to 24h of the inoculated cells, pWS46, pGY32, pGY102, pSZ69, pSZ83 and pSZ84 were added to the first group, and pWS46, pGY32, pGY102, pSZ69 and pSZ92 (P) were added to the second group _U6 -gRNA1 (IL 1 RN) -pA) and pSZ93 (P) _U6 gRNA2 (IL 1 RN) -pA), and in the third group pWS46, pGY32, pGY102, pSZ69, pSZ105 (P) were added _U6 -gRNA1 (RHOXF 2) -pA) and pSZ106 (P) _U6 -gRNA2 (RHOXF 2) -pA), in the fourth group pWS46, pGY32, pGY102, pSZ69, pSZ103 (P) were added _U6 -gRNA1 (TTN) -pA) and pSZ104 (P) _U6 -gRNA2 (TTN) -pA), in the fifth group pWS46, pGY32, pGY102, pSZ69, pSZ83, pSZ84, pSZ92 (P) were added _U6 -gRNA1(IL1RN)-pA)、pSZ93(P _U6 -gRNA2(IL1RN)-pA)、pSZ105(P _U6 -gRNA1(RHOXF2)-pA)、pSZ106(P _U6 -gRNA2(RHOXF2)-pA)、pSZ103(P _U6 -gRNA1 (TTN) -pA) and pSZ104 (P) _U6 -gRNA2 (TTN) -pA), mixed with PEI transfection reagent and serum-free DMEM, left to stand at room temperature for 15min and added to 24-well culture plates. Wherein the total preparation volume of each hole is 50 mu L, and the mass ratio of the plasmid to the PEI is 1. 6h after transfection, the cells were cultured in 500. Mu.L of DMEM medium containing 10% FBS.

And fourthly, illuminating. After the liquid is changed for 14-18h, the illumination group is placed at the wavelength of 730nm and the illumination intensity of 1.5mW/cm ² The LEDs respectively illuminate for 6h/d, and the illumination is immediately finishedThe cells were cultured in the dark, and the dark group was cultured in the dark.

The result shows that the genome transcription device based on the CRISPR/Cas9 system and regulated by far-red light can simultaneously and efficiently activate a plurality of endogenous genes. Experimental data are detailed in figure 14 of the specification, all presented as n =3 independent replication experiments.

TABLE 1 plasmid construction Table

1, SEQ ID NO.1: bldD binding site (whiG) nucleotide sequence

CTCACGCTACGCTCA

SEQ ID NO.2: nucleotide sequence of TATABox

TAGAGGGTATATAATGGAAGCTCG

SEQ ID NO.3: cytomegalovirus minimal promoter CMV _min Nucleotide sequence of (A)

CCTGCAGGTCGAGCTCGGTACCCGGGTCGAGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCG

SEQ ID NO.4: cytomegalovirus minimal promoter CMV _min Mutant CMV _min3G <xnotran> AATGTCGAGGTAGGCGTGTACGGTGGGCGCCTATAAAAGCAGAGCTCGTTAGTGAACCGTCAGATCGCCTGGAGCAATTCCACAACACTTTTGTCTTATACTTGGATCACC </xnotran>

SEQ ID NO.5：P _FRL1a: pA-3*whiG-P _hCMVmin

CAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAACCTCACGCTACGCTCACTCACGCTACGCTCACTCACGCTACGCTCACCTGCAGGTCGAGCTCGGTACCCGGGTCGAGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCG

SEQ ID NO.6：P _FRL2a: 3*whiG-P _hCMVmin

CTCACGCTACGCTCACTCACGCTACGCTCACTCACGCTACGCTCACCTGCAGGTCGAGCTCGGTACCCGGGTCGAGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCG

SEQ ID NO.7：P _FRL3a: 1*whiG-P _hCMVmin

CTCACGCTACGCTCACCTGCAGGTCGAGCTCGGTACCCGGGTCGAGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCG

SEQ ID NO.8：P _FRL1b: 2*whiG-P _hCMVmin3G

CTCACGCTACGCTCACTCACGCTACGCTCACCTGCAGGATGTCGAGGTAGGCGTGTACGGTGGGCGCCTATAAAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGCAATTCCACAACACTTTTGTCTTATACTTGGATCACC

SEQ ID NO.9：P _FRL2b: 3*whiG-P _hCMVmin3G

CTCACGCTACGCTCACTCACGCTACGCTCACTCACGCTACGCTCACCTGCAGGATGTCGAGGTAGGCGTGTACGGTGGGCGCCTATAAAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGCAATTCCACAACACTTTTGTCTTATACTTGGATCACC

SEQ ID NO.10: <xnotran> Cas9 (D10A, H840A) dCas9 MYPYDVPDYASPKKKRKVEASDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSPKKKRKVEAS </xnotran>

SEQ ID No.11: herpes simplex virion protein VP16 transcriptional activation domain and 4 copies of the transcriptional activation domain VP64

MGSGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLIN

SEQ ID No.12: NF-KB p65 subunit transcriptional activation domain

GSPSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTPGPPQSL

SEQ ID NO.13: coat protein MS2

MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYSA

SEQ ID No.14: VP64-p65-Rta fusion protein

GSGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLYIDSSGSPKKKRKVGPSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTPGPPQSLGSGSGSRDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWANRPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKALREMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEILDTFLNDECLLHAMHISTGLSIFDTSLF

SEQ ID NO.15: heat shock transcription factor HSF1 transcriptional activation domain

SAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSEFQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDEDFSSIADMDFSALLSQISSSGQGGGGSGFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPRPPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAEDPTISLLTGSEPPKAKDPTVS

SEQ ID NO.16：FGTA1:MS2-Linker-NLS-VP64

MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYSAGGGGSGGGGSGGGGSGPKKKRKVAAAMGSGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLIN

SEQ ID NO.17：FGTA2:MS2-Linker-NLS-VPR

MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYSAGGGGSGGGGSGGGGSGPKKKRKVAAAGSGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLYIDSSGSPKKKRKVGPSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTPGPPQSLGSGSGSRDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWANRPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKALREMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEILDTFLNDECLLHAMHISTGLSIFDTSLF

SEQ ID NO.18：FGTA3:MS2-Linker-NLS-p65-HSF1-VP64

MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYSAGGGGSGGGGSGGGGSGPKKKRKVAAAGSPSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTPGPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSEFQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDEDFSSIADMDFSALLSQISSSGQGGGGSGFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPRPPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAEDPTISLLTGSEPPKAKDPTVSMGSGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLIN

SEQ ID NO.19：FGTA4:MS2-Linker-NLS-p65-HSF1

MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYSAGGGGSGGGGSGGGGSGPKKKRKVAAAGSPSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTPGPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSEFQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDEDFSSIADMDFSALLSQISSSGQGGGGSGFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPRPPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAEDPTISLLTGSEPPKAKDPTVS

SEQ ID NO.20, the far-red processor (BldD) amino acid sequence

MASPKKKRKVEASSSEYAKQLGAKLRAIRTQQGLSLHGVEEKSQGRWKAVVVGSYERGDRAVTVQRLAELADFYGVPVQELLPGTTPGGAAEPPPKLVLDLERLAHVPQEKAGPLQRYAATIQSQRGDYNGKVLSIRQDDLRTLAVIYDQSPSVLTEQLISWGVLDADARRAVAHEEN

SEQ ID NO.21, amino acid sequence of Nuclear Localization Signal (NLS)

TSPKKKRKVEDTS

SEQ ID NO.22, amino acid sequence of connecting functional peptide (Linker)

ASGSGGG

SEQ ID NO.23 sequence of bacterial photosensitive diguanylate cyclase (BphS)

MARGCLMTISGGTFDPSICEMEPIATPGAIQPHGALMTARADSGRVAHASVNLGEILGLPAASVLGAPIGEVIGRVNEILLREARRSGSETPETIGSFRRSDGQLLHLHAFQSGDYMCLDIEPVRDEDGRLPPGARQSVIETFSSAMTQVELCELAVHGLQLVLGYDRVMAYRFGADGHGEVIAERRRQDLEPYLGLHYPASDIPQIARALYLRQRVGAIADACYRPVPLLGHPELDDGKPLDLTHSSLRSVSPVHLDYMQNMNTAASLTIGLADGDRLWGMLVCHNTTPRIAGPEWRAAAGMIGQVVSLLLSRLGEVENAAETLARQSTLSTLVERLSTGDTLAAAFVAADQLILDLVGASAAVVRLAGQELHFGRTPPVDAMQKVLDSLGRPSPLEVLSLDDVTLRHPELPELLAAGSGILLLPLTSGDGDLIAWFRPEHVQTITWGGNPAEHGTWNPATQRMRPRASFDAWKETVTGRSLPWTSAERNCARELGEAIAAEMAQRTRAEELERVAMVDSLTRLWNRLGIETLLKREWEYATRKNSPISIVMIDFDNFKQINDQHGHLVGDEVLQGSARLIISVLASYDILGRWGGDEFMLILPGSGREQTAVLLERIQATIAQNPVPTSAGPMAISLSMGGVSVFTNQGEALQYWVEQADNQLMKVKRLGKGNFQLAEYHHHHHH

Amino acid sequence of fusion protein p65-VP64-BldD with downstream response of SEQ ID NO.24

ATMPSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTPGPPQSLMGSGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLINASGSGGGGDVMASPKKKRKVEASSSEYAKQLGAKLRAIRTQQGLSLHGVEEKSQGRWKAVVVGSYERGDRAVTVQRLAELADFYGVPVQELLPGTTPGGAAEPPPKLVLDLERLAHVPQEKAGPLQRYAATIQSQRGDYNGKVLSIRQDDLRTLAVIYDQSPSVLTEQLISWGVLDADARRAVAHEEN

The protection content of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected.

SEQUENCE LISTING

<110> university of east China

<120> genome transcription device based on CRISPR/Cas9 system and regulated by far-red light, construction method and application

<160> 67

<170> PatentIn version 3.3

<210> 1

<211> 15

<212> DNA

<213> Artificial sequence

<400> 1

ctcacgctac gctca 15

<210> 2

<211> 24

<212> DNA

<213> Artificial sequence

<400> 2

tagagggtat ataatggaag ctcg 24

<210> 3

<211> 156

<212> DNA

<213> Artificial sequence

<400> 3

cctgcaggtc gagctcggta cccgggtcga gtaggcgtgt acggtgggag gcctatataa 60

gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc 120

tccatagaag acaccgggac cgatccagcc tccgcg 156

<210> 4

<211> 111

<212> DNA

<213> Artificial sequence

<400> 4

aatgtcgagg taggcgtgta cggtgggcgc ctataaaagc agagctcgtt agtgaaccgt 60

cagatcgcct ggagcaattc cacaacactt ttgtcttata cttggatcac c 111

<210> 5

<211> 397

<212> DNA

<213> Artificial sequence

<400> 5

cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa 60

aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca 120

ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt 180

gggaggtttt ttaaacctca cgctacgctc actcacgcta cgctcactca cgctacgctc 240

acctgcaggt cgagctcggt acccgggtcg agtaggcgtg tacggtggga ggcctatata 300

agcagagctc gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac 360

ctccatagaa gacaccggga ccgatccagc ctccgcg 397

<210> 6

<211> 201

<212> DNA

<213> Artificial sequence

<400> 6

ctcacgctac gctcactcac gctacgctca ctcacgctac gctcacctgc aggtcgagct 60

cggtacccgg gtcgagtagg cgtgtacggt gggaggccta tataagcaga gctcgtttag 120

tgaaccgtca gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagacacc 180

gggaccgatc cagcctccgc g 201

<210> 7

<211> 171

<212> DNA

<213> Artificial sequence

<400> 7

ctcacgctac gctcacctgc aggtcgagct cggtacccgg gtcgagtagg cgtgtacggt 60

gggaggccta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg agacgccatc 120

cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgc g 171

<210> 8

<211> 149

<212> DNA

<213> Artificial sequence

<400> 8

ctcacgctac gctcactcac gctacgctca cctgcaggat gtcgaggtag gcgtgtacgg 60

tgggcgccta taaaagcaga gctcgtttag tgaaccgtca gatcgcctgg agcaattcca 120

caacactttt gtcttatact tggatcacc 149

<210> 9

<211> 164

<212> DNA

<213> Artificial sequence

<400> 9

ctcacgctac gctcactcac gctacgctca ctcacgctac gctcacctgc aggatgtcga 60

ggtaggcgtg tacggtgggc gcctataaaa gcagagctcg tttagtgaac cgtcagatcg 120

cctggagcaa ttccacaaca cttttgtctt atacttggat cacc 164

<210> 10

<211> 1399

<212> PRT

<213> Artificial sequence

<400> 10

Met Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Pro Lys Lys Lys Arg

1 5 10 15

Lys Val Glu Ala Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly

20 25 30

Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro

35 40 45

Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys

50 55 60

Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu

65 70 75 80

Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys

85 90 95

Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys

100 105 110

Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu

115 120 125

Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp

130 135 140

Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys

145 150 155 160

Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu

165 170 175

Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly

180 185 190

Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu

195 200 205

Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser

210 215 220

Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg

225 230 235 240

Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly

245 250 255

Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe

260 265 270

Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys

275 280 285

Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp

290 295 300

Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile

305 310 315 320

Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro

325 330 335

Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu

340 345 350

Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys

355 360 365

Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp

370 375 380

Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu

385 390 395 400

Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu

405 410 415

Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His

420 425 430

Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp

435 440 445

Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu

450 455 460

Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser

465 470 475 480

Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp

485 490 495

Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile

500 505 510

Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu

515 520 525

Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu

530 535 540

Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu

545 550 555 560

Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn

565 570 575

Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile

580 585 590

Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn

595 600 605

Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys

610 615 620

Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val

625 630 635 640

Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu

645 650 655

Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys

660 665 670

Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn

675 680 685

Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys

690 695 700

Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp

705 710 715 720

Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln

725 730 735

Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala

740 745 750

Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val

755 760 765

Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala

770 775 780

Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg

785 790 795 800

Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu

805 810 815

Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr

820 825 830

Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu

835 840 845

Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln

850 855 860

Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser

865 870 875 880

Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val

885 890 895

Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile

900 905 910

Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu

915 920 925

Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr

930 935 940

Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn

945 950 955 960

Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile

965 970 975

Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe

980 985 990

Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr

995 1000 1005

Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys

1010 1015 1020

Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val

1025 1030 1035

Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr

1040 1045 1050

Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr

1055 1060 1065

Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile

1070 1075 1080

Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg

1085 1090 1095

Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn

1100 1105 1110

Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu

1115 1120 1125

Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys

1130 1135 1140

Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr

1145 1150 1155

Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys

1160 1165 1170

Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile

1175 1180 1185

Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu

1190 1195 1200

Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu

1205 1210 1215

Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met

1220 1225 1230

Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu

1235 1240 1245

Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu

1250 1255 1260

Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe

1265 1270 1275

Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile

1280 1285 1290

Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp

1295 1300 1305

Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg

1310 1315 1320

Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu

1325 1330 1335

Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg

1340 1345 1350

Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile

1355 1360 1365

His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser

1370 1375 1380

Gln Leu Gly Gly Asp Ser Pro Lys Lys Lys Arg Lys Val Glu Ala

1385 1390 1395

Ser

<210> 11

<211> 58

<212> PRT

<213> Artificial sequence

<400> 11

Met Gly Ser Gly Arg Ala Asp Ala Leu Asp Asp Phe Asp Leu Asp Met

1 5 10 15

Leu Gly Ser Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Ser

20 25 30

Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu

35 40 45

Asp Asp Phe Asp Leu Asp Met Leu Ile Asn

50 55

<210> 12

<211> 53

<212> PRT

<213> Artificial sequence

<400> 12

Gly Ser Pro Ser Gly Gln Ile Ser Asn Gln Ala Leu Ala Leu Ala Pro

1 5 10 15

Ser Ser Ala Pro Val Leu Ala Gln Thr Met Val Pro Ser Ser Ala Met

20 25 30

Val Pro Leu Ala Gln Pro Pro Ala Pro Ala Pro Val Leu Thr Pro Gly

35 40 45

Pro Pro Gln Ser Leu

50

<210> 13

<211> 132

<212> PRT

<213> Artificial sequence

<400> 13

Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr

1 5 10 15

Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu

20 25 30

Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser

35 40 45

Val Arg Gln Ser Ser Ala Gln Lys Arg Lys Tyr Thr Ile Lys Val Glu

50 55 60

Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val

65 70 75 80

Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro Ile Phe

85 90 95

Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln Gly Leu

100 105 110

Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser Gly

115 120 125

Ile Tyr Ser Ala

130

<210> 14

<211> 317

<212> PRT

<213> Artificial sequence

<400> 14

Gly Ser Gly Arg Ala Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu

1 5 10 15

Gly Ser Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Ser Asp

20 25 30

Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp

35 40 45

Asp Phe Asp Leu Asp Met Leu Tyr Ile Asp Ser Ser Gly Ser Pro Lys

50 55 60

Lys Lys Arg Lys Val Gly Pro Ser Gly Gln Ile Ser Asn Gln Ala Leu

65 70 75 80

Ala Leu Ala Pro Ser Ser Ala Pro Val Leu Ala Gln Thr Met Val Pro

85 90 95

Ser Ser Ala Met Val Pro Leu Ala Gln Pro Pro Ala Pro Ala Pro Val

100 105 110

Leu Thr Pro Gly Pro Pro Gln Ser Leu Gly Ser Gly Ser Gly Ser Arg

115 120 125

Asp Ser Arg Glu Gly Met Phe Leu Pro Lys Pro Glu Ala Gly Ser Ala

130 135 140

Ile Ser Asp Val Phe Glu Gly Arg Glu Val Cys Gln Pro Lys Arg Ile

145 150 155 160

Arg Pro Phe His Pro Pro Gly Ser Pro Trp Ala Asn Arg Pro Leu Pro

165 170 175

Ala Ser Leu Ala Pro Thr Pro Thr Gly Pro Val His Glu Pro Val Gly

180 185 190

Ser Leu Thr Pro Ala Pro Val Pro Gln Pro Leu Asp Pro Ala Pro Ala

195 200 205

Val Thr Pro Glu Ala Ser His Leu Leu Glu Asp Pro Asp Glu Glu Thr

210 215 220

Ser Gln Ala Val Lys Ala Leu Arg Glu Met Ala Asp Thr Val Ile Pro

225 230 235 240

Gln Lys Glu Glu Ala Ala Ile Cys Gly Gln Met Asp Leu Ser His Pro

245 250 255

Pro Pro Arg Gly His Leu Asp Glu Leu Thr Thr Thr Leu Glu Ser Met

260 265 270

Thr Glu Asp Leu Asn Leu Asp Ser Pro Leu Thr Pro Glu Leu Asn Glu

275 280 285

Ile Leu Asp Thr Phe Leu Asn Asp Glu Cys Leu Leu His Ala Met His

290 295 300

Ile Ser Thr Gly Leu Ser Ile Phe Asp Thr Ser Leu Phe

305 310 315

<210> 15

<211> 262

<212> PRT

<213> Artificial sequence

<400> 15

Ser Ala Pro Val Pro Lys Ser Thr Gln Ala Gly Glu Gly Thr Leu Ser

1 5 10 15

Glu Ala Leu Leu His Leu Gln Phe Asp Ala Asp Glu Asp Leu Gly Ala

20 25 30

Leu Leu Gly Asn Ser Thr Asp Pro Gly Val Phe Thr Asp Leu Ala Ser

35 40 45

Val Asp Asn Ser Glu Phe Gln Gln Leu Leu Asn Gln Gly Val Ser Met

50 55 60

Ser His Ser Thr Ala Glu Pro Met Leu Met Glu Tyr Pro Glu Ala Ile

65 70 75 80

Thr Arg Leu Val Thr Gly Ser Gln Arg Pro Pro Asp Pro Ala Pro Thr

85 90 95

Pro Leu Gly Thr Ser Gly Leu Pro Asn Gly Leu Ser Gly Asp Glu Asp

100 105 110

Phe Ser Ser Ile Ala Asp Met Asp Phe Ser Ala Leu Leu Ser Gln Ile

115 120 125

Ser Ser Ser Gly Gln Gly Gly Gly Gly Ser Gly Phe Ser Val Asp Thr

130 135 140

Ser Ala Leu Leu Asp Leu Phe Ser Pro Ser Val Thr Val Pro Asp Met

145 150 155 160

Ser Leu Pro Asp Leu Asp Ser Ser Leu Ala Ser Ile Gln Glu Leu Leu

165 170 175

Ser Pro Gln Glu Pro Pro Arg Pro Pro Glu Ala Glu Asn Ser Ser Pro

180 185 190

Asp Ser Gly Lys Gln Leu Val His Tyr Thr Ala Gln Pro Leu Phe Leu

195 200 205

Leu Asp Pro Gly Ser Val Asp Thr Gly Ser Asn Asp Leu Pro Val Leu

210 215 220

Phe Glu Leu Gly Glu Gly Ser Tyr Phe Ser Glu Gly Asp Gly Phe Ala

225 230 235 240

Glu Asp Pro Thr Ile Ser Leu Leu Thr Gly Ser Glu Pro Pro Lys Ala

245 250 255

Lys Asp Pro Thr Val Ser

260

<210> 16

<211> 216

<212> PRT

<213> Artificial sequence

<400> 16

Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr

1 5 10 15

Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu

20 25 30

Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser

35 40 45

Val Arg Gln Ser Ser Ala Gln Lys Arg Lys Tyr Thr Ile Lys Val Glu

50 55 60

Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val

65 70 75 80

Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro Ile Phe

85 90 95

Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln Gly Leu

100 105 110

Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser Gly

115 120 125

Ile Tyr Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Pro Lys Lys Lys Arg Lys Val Ala Ala Ala Met Gly

145 150 155 160

Ser Gly Arg Ala Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly

165 170 175

Ser Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala

180 185 190

Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp Asp

195 200 205

Phe Asp Leu Asp Met Leu Ile Asn

210 215

<210> 17

<211> 475

<212> PRT

<213> Artificial sequence

<400> 17

Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr

1 5 10 15

Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu

20 25 30

Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser

35 40 45

Val Arg Gln Ser Ser Ala Gln Lys Arg Lys Tyr Thr Ile Lys Val Glu

50 55 60

Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val

65 70 75 80

Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro Ile Phe

85 90 95

Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln Gly Leu

100 105 110

Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser Gly

115 120 125

Ile Tyr Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Pro Lys Lys Lys Arg Lys Val Ala Ala Ala Gly Ser

145 150 155 160

Gly Arg Ala Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Ser

165 170 175

Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu

180 185 190

Asp Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe

195 200 205

Asp Leu Asp Met Leu Tyr Ile Asp Ser Ser Gly Ser Pro Lys Lys Lys

210 215 220

Arg Lys Val Gly Pro Ser Gly Gln Ile Ser Asn Gln Ala Leu Ala Leu

225 230 235 240

Ala Pro Ser Ser Ala Pro Val Leu Ala Gln Thr Met Val Pro Ser Ser

245 250 255

Ala Met Val Pro Leu Ala Gln Pro Pro Ala Pro Ala Pro Val Leu Thr

260 265 270

Pro Gly Pro Pro Gln Ser Leu Gly Ser Gly Ser Gly Ser Arg Asp Ser

275 280 285

Arg Glu Gly Met Phe Leu Pro Lys Pro Glu Ala Gly Ser Ala Ile Ser

290 295 300

Asp Val Phe Glu Gly Arg Glu Val Cys Gln Pro Lys Arg Ile Arg Pro

305 310 315 320

Phe His Pro Pro Gly Ser Pro Trp Ala Asn Arg Pro Leu Pro Ala Ser

325 330 335

Leu Ala Pro Thr Pro Thr Gly Pro Val His Glu Pro Val Gly Ser Leu

340 345 350

Thr Pro Ala Pro Val Pro Gln Pro Leu Asp Pro Ala Pro Ala Val Thr

355 360 365

Pro Glu Ala Ser His Leu Leu Glu Asp Pro Asp Glu Glu Thr Ser Gln

370 375 380

Ala Val Lys Ala Leu Arg Glu Met Ala Asp Thr Val Ile Pro Gln Lys

385 390 395 400

Glu Glu Ala Ala Ile Cys Gly Gln Met Asp Leu Ser His Pro Pro Pro

405 410 415

Arg Gly His Leu Asp Glu Leu Thr Thr Thr Leu Glu Ser Met Thr Glu

420 425 430

Asp Leu Asn Leu Asp Ser Pro Leu Thr Pro Glu Leu Asn Glu Ile Leu

435 440 445

Asp Thr Phe Leu Asn Asp Glu Cys Leu Leu His Ala Met His Ile Ser

450 455 460

Thr Gly Leu Ser Ile Phe Asp Thr Ser Leu Phe

465 470 475

<210> 18

<211> 531

<212> PRT

<213> Artificial sequence

<400> 18

Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr

1 5 10 15

Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu

20 25 30

Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser

35 40 45

Val Arg Gln Ser Ser Ala Gln Lys Arg Lys Tyr Thr Ile Lys Val Glu

50 55 60

Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val

65 70 75 80

Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro Ile Phe

85 90 95

Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln Gly Leu

100 105 110

Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser Gly

115 120 125

Ile Tyr Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Pro Lys Lys Lys Arg Lys Val Ala Ala Ala Gly Ser

145 150 155 160

Pro Ser Gly Gln Ile Ser Asn Gln Ala Leu Ala Leu Ala Pro Ser Ser

165 170 175

Ala Pro Val Leu Ala Gln Thr Met Val Pro Ser Ser Ala Met Val Pro

180 185 190

Leu Ala Gln Pro Pro Ala Pro Ala Pro Val Leu Thr Pro Gly Pro Pro

195 200 205

Gln Ser Leu Ser Ala Pro Val Pro Lys Ser Thr Gln Ala Gly Glu Gly

210 215 220

Thr Leu Ser Glu Ala Leu Leu His Leu Gln Phe Asp Ala Asp Glu Asp

225 230 235 240

Leu Gly Ala Leu Leu Gly Asn Ser Thr Asp Pro Gly Val Phe Thr Asp

245 250 255

Leu Ala Ser Val Asp Asn Ser Glu Phe Gln Gln Leu Leu Asn Gln Gly

260 265 270

Val Ser Met Ser His Ser Thr Ala Glu Pro Met Leu Met Glu Tyr Pro

275 280 285

Glu Ala Ile Thr Arg Leu Val Thr Gly Ser Gln Arg Pro Pro Asp Pro

290 295 300

Ala Pro Thr Pro Leu Gly Thr Ser Gly Leu Pro Asn Gly Leu Ser Gly

305 310 315 320

Asp Glu Asp Phe Ser Ser Ile Ala Asp Met Asp Phe Ser Ala Leu Leu

325 330 335

Ser Gln Ile Ser Ser Ser Gly Gln Gly Gly Gly Gly Ser Gly Phe Ser

340 345 350

Val Asp Thr Ser Ala Leu Leu Asp Leu Phe Ser Pro Ser Val Thr Val

355 360 365

Pro Asp Met Ser Leu Pro Asp Leu Asp Ser Ser Leu Ala Ser Ile Gln

370 375 380

Glu Leu Leu Ser Pro Gln Glu Pro Pro Arg Pro Pro Glu Ala Glu Asn

385 390 395 400

Ser Ser Pro Asp Ser Gly Lys Gln Leu Val His Tyr Thr Ala Gln Pro

405 410 415

Leu Phe Leu Leu Asp Pro Gly Ser Val Asp Thr Gly Ser Asn Asp Leu

420 425 430

Pro Val Leu Phe Glu Leu Gly Glu Gly Ser Tyr Phe Ser Glu Gly Asp

435 440 445

Gly Phe Ala Glu Asp Pro Thr Ile Ser Leu Leu Thr Gly Ser Glu Pro

450 455 460

Pro Lys Ala Lys Asp Pro Thr Val Ser Met Gly Ser Gly Arg Ala Asp

465 470 475 480

Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp

485 490 495

Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe Asp

500 505 510

Leu Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe Asp Leu Asp Met

515 520 525

Leu Ile Asn

530

<210> 19

<211> 473

<212> PRT

<213> Artificial sequence

<400> 19

Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr

1 5 10 15

Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu

20 25 30

Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser

35 40 45

Val Arg Gln Ser Ser Ala Gln Lys Arg Lys Tyr Thr Ile Lys Val Glu

50 55 60

Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val

65 70 75 80

Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro Ile Phe

85 90 95

Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln Gly Leu

100 105 110

Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser Gly

115 120 125

Ile Tyr Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Pro Lys Lys Lys Arg Lys Val Ala Ala Ala Gly Ser

145 150 155 160

Pro Ser Gly Gln Ile Ser Asn Gln Ala Leu Ala Leu Ala Pro Ser Ser

165 170 175

Ala Pro Val Leu Ala Gln Thr Met Val Pro Ser Ser Ala Met Val Pro

180 185 190

Leu Ala Gln Pro Pro Ala Pro Ala Pro Val Leu Thr Pro Gly Pro Pro

195 200 205

Gln Ser Leu Ser Ala Pro Val Pro Lys Ser Thr Gln Ala Gly Glu Gly

210 215 220

Thr Leu Ser Glu Ala Leu Leu His Leu Gln Phe Asp Ala Asp Glu Asp

225 230 235 240

Leu Gly Ala Leu Leu Gly Asn Ser Thr Asp Pro Gly Val Phe Thr Asp

245 250 255

Leu Ala Ser Val Asp Asn Ser Glu Phe Gln Gln Leu Leu Asn Gln Gly

260 265 270

Val Ser Met Ser His Ser Thr Ala Glu Pro Met Leu Met Glu Tyr Pro

275 280 285

Glu Ala Ile Thr Arg Leu Val Thr Gly Ser Gln Arg Pro Pro Asp Pro

290 295 300

Ala Pro Thr Pro Leu Gly Thr Ser Gly Leu Pro Asn Gly Leu Ser Gly

305 310 315 320

Asp Glu Asp Phe Ser Ser Ile Ala Asp Met Asp Phe Ser Ala Leu Leu

325 330 335

Ser Gln Ile Ser Ser Ser Gly Gln Gly Gly Gly Gly Ser Gly Phe Ser

340 345 350

Val Asp Thr Ser Ala Leu Leu Asp Leu Phe Ser Pro Ser Val Thr Val

355 360 365

Pro Asp Met Ser Leu Pro Asp Leu Asp Ser Ser Leu Ala Ser Ile Gln

370 375 380

Glu Leu Leu Ser Pro Gln Glu Pro Pro Arg Pro Pro Glu Ala Glu Asn

385 390 395 400

Ser Ser Pro Asp Ser Gly Lys Gln Leu Val His Tyr Thr Ala Gln Pro

405 410 415

Leu Phe Leu Leu Asp Pro Gly Ser Val Asp Thr Gly Ser Asn Asp Leu

420 425 430

Pro Val Leu Phe Glu Leu Gly Glu Gly Ser Tyr Phe Ser Glu Gly Asp

435 440 445

Gly Phe Ala Glu Asp Pro Thr Ile Ser Leu Leu Thr Gly Ser Glu Pro

450 455 460

Pro Lys Ala Lys Asp Pro Thr Val Ser

465 470

<210> 20

<211> 178

<212> PRT

<213> Artificial sequence

<400> 20

Met Ala Ser Pro Lys Lys Lys Arg Lys Val Glu Ala Ser Ser Ser Glu

1 5 10 15

Tyr Ala Lys Gln Leu Gly Ala Lys Leu Arg Ala Ile Arg Thr Gln Gln

20 25 30

Gly Leu Ser Leu His Gly Val Glu Glu Lys Ser Gln Gly Arg Trp Lys

35 40 45

Ala Val Val Val Gly Ser Tyr Glu Arg Gly Asp Arg Ala Val Thr Val

50 55 60

Gln Arg Leu Ala Glu Leu Ala Asp Phe Tyr Gly Val Pro Val Gln Glu

65 70 75 80

Leu Leu Pro Gly Thr Thr Pro Gly Gly Ala Ala Glu Pro Pro Pro Lys

85 90 95

Leu Val Leu Asp Leu Glu Arg Leu Ala His Val Pro Gln Glu Lys Ala

100 105 110

Gly Pro Leu Gln Arg Tyr Ala Ala Thr Ile Gln Ser Gln Arg Gly Asp

115 120 125

Tyr Asn Gly Lys Val Leu Ser Ile Arg Gln Asp Asp Leu Arg Thr Leu

130 135 140

Ala Val Ile Tyr Asp Gln Ser Pro Ser Val Leu Thr Glu Gln Leu Ile

145 150 155 160

Ser Trp Gly Val Leu Asp Ala Asp Ala Arg Arg Ala Val Ala His Glu

165 170 175

Glu Asn

<210> 21

<211> 13

<212> PRT

<213> Artificial sequence

<400> 21

Thr Ser Pro Lys Lys Lys Arg Lys Val Glu Asp Thr Ser

1 5 10

<210> 22

<211> 7

<212> PRT

<213> Artificial sequence

<400> 22

Ala Ser Gly Ser Gly Gly Gly

1 5

<210> 23

<211> 687

<212> PRT

<213> Artificial sequence

<400> 23

Met Ala Arg Gly Cys Leu Met Thr Ile Ser Gly Gly Thr Phe Asp Pro

1 5 10 15

Ser Ile Cys Glu Met Glu Pro Ile Ala Thr Pro Gly Ala Ile Gln Pro

20 25 30

His Gly Ala Leu Met Thr Ala Arg Ala Asp Ser Gly Arg Val Ala His

35 40 45

Ala Ser Val Asn Leu Gly Glu Ile Leu Gly Leu Pro Ala Ala Ser Val

50 55 60

Leu Gly Ala Pro Ile Gly Glu Val Ile Gly Arg Val Asn Glu Ile Leu

65 70 75 80

Leu Arg Glu Ala Arg Arg Ser Gly Ser Glu Thr Pro Glu Thr Ile Gly

85 90 95

Ser Phe Arg Arg Ser Asp Gly Gln Leu Leu His Leu His Ala Phe Gln

100 105 110

Ser Gly Asp Tyr Met Cys Leu Asp Ile Glu Pro Val Arg Asp Glu Asp

115 120 125

Gly Arg Leu Pro Pro Gly Ala Arg Gln Ser Val Ile Glu Thr Phe Ser

130 135 140

Ser Ala Met Thr Gln Val Glu Leu Cys Glu Leu Ala Val His Gly Leu

145 150 155 160

Gln Leu Val Leu Gly Tyr Asp Arg Val Met Ala Tyr Arg Phe Gly Ala

165 170 175

Asp Gly His Gly Glu Val Ile Ala Glu Arg Arg Arg Gln Asp Leu Glu

180 185 190

Pro Tyr Leu Gly Leu His Tyr Pro Ala Ser Asp Ile Pro Gln Ile Ala

195 200 205

Arg Ala Leu Tyr Leu Arg Gln Arg Val Gly Ala Ile Ala Asp Ala Cys

210 215 220

Tyr Arg Pro Val Pro Leu Leu Gly His Pro Glu Leu Asp Asp Gly Lys

225 230 235 240

Pro Leu Asp Leu Thr His Ser Ser Leu Arg Ser Val Ser Pro Val His

245 250 255

Leu Asp Tyr Met Gln Asn Met Asn Thr Ala Ala Ser Leu Thr Ile Gly

260 265 270

Leu Ala Asp Gly Asp Arg Leu Trp Gly Met Leu Val Cys His Asn Thr

275 280 285

Thr Pro Arg Ile Ala Gly Pro Glu Trp Arg Ala Ala Ala Gly Met Ile

290 295 300

Gly Gln Val Val Ser Leu Leu Leu Ser Arg Leu Gly Glu Val Glu Asn

305 310 315 320

Ala Ala Glu Thr Leu Ala Arg Gln Ser Thr Leu Ser Thr Leu Val Glu

325 330 335

Arg Leu Ser Thr Gly Asp Thr Leu Ala Ala Ala Phe Val Ala Ala Asp

340 345 350

Gln Leu Ile Leu Asp Leu Val Gly Ala Ser Ala Ala Val Val Arg Leu

355 360 365

Ala Gly Gln Glu Leu His Phe Gly Arg Thr Pro Pro Val Asp Ala Met

370 375 380

Gln Lys Val Leu Asp Ser Leu Gly Arg Pro Ser Pro Leu Glu Val Leu

385 390 395 400

Ser Leu Asp Asp Val Thr Leu Arg His Pro Glu Leu Pro Glu Leu Leu

405 410 415

Ala Ala Gly Ser Gly Ile Leu Leu Leu Pro Leu Thr Ser Gly Asp Gly

420 425 430

Asp Leu Ile Ala Trp Phe Arg Pro Glu His Val Gln Thr Ile Thr Trp

435 440 445

Gly Gly Asn Pro Ala Glu His Gly Thr Trp Asn Pro Ala Thr Gln Arg

450 455 460

Met Arg Pro Arg Ala Ser Phe Asp Ala Trp Lys Glu Thr Val Thr Gly

465 470 475 480

Arg Ser Leu Pro Trp Thr Ser Ala Glu Arg Asn Cys Ala Arg Glu Leu

485 490 495

Gly Glu Ala Ile Ala Ala Glu Met Ala Gln Arg Thr Arg Ala Glu Glu

500 505 510

Leu Glu Arg Val Ala Met Val Asp Ser Leu Thr Arg Leu Trp Asn Arg

515 520 525

Leu Gly Ile Glu Thr Leu Leu Lys Arg Glu Trp Glu Tyr Ala Thr Arg

530 535 540

Lys Asn Ser Pro Ile Ser Ile Val Met Ile Asp Phe Asp Asn Phe Lys

545 550 555 560

Gln Ile Asn Asp Gln His Gly His Leu Val Gly Asp Glu Val Leu Gln

565 570 575

Gly Ser Ala Arg Leu Ile Ile Ser Val Leu Ala Ser Tyr Asp Ile Leu

580 585 590

Gly Arg Trp Gly Gly Asp Glu Phe Met Leu Ile Leu Pro Gly Ser Gly

595 600 605

Arg Glu Gln Thr Ala Val Leu Leu Glu Arg Ile Gln Ala Thr Ile Ala

610 615 620

Gln Asn Pro Val Pro Thr Ser Ala Gly Pro Met Ala Ile Ser Leu Ser

625 630 635 640

Met Gly Gly Val Ser Val Phe Thr Asn Gln Gly Glu Ala Leu Gln Tyr

645 650 655

Trp Val Glu Gln Ala Asp Asn Gln Leu Met Lys Val Lys Arg Leu Gly

660 665 670

Lys Gly Asn Phe Gln Leu Ala Glu Tyr His His His His His His

675 680 685

<210> 24

<211> 300

<212> PRT

<213> Artificial sequence

<400> 24

Ala Thr Met Pro Ser Gly Gln Ile Ser Asn Gln Ala Leu Ala Leu Ala

1 5 10 15

Pro Ser Ser Ala Pro Val Leu Ala Gln Thr Met Val Pro Ser Ser Ala

20 25 30

Met Val Pro Leu Ala Gln Pro Pro Ala Pro Ala Pro Val Leu Thr Pro

35 40 45

Gly Pro Pro Gln Ser Leu Met Gly Ser Gly Arg Ala Asp Ala Leu Asp

50 55 60

Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe Asp

65 70 75 80

Leu Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe Asp Leu Asp Met

85 90 95

Leu Gly Ser Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Ile Asn

100 105 110

Ala Ser Gly Ser Gly Gly Gly Gly Asp Val Met Ala Ser Pro Lys Lys

115 120 125

Lys Arg Lys Val Glu Ala Ser Ser Ser Glu Tyr Ala Lys Gln Leu Gly

130 135 140

Ala Lys Leu Arg Ala Ile Arg Thr Gln Gln Gly Leu Ser Leu His Gly

145 150 155 160

Val Glu Glu Lys Ser Gln Gly Arg Trp Lys Ala Val Val Val Gly Ser

165 170 175

Tyr Glu Arg Gly Asp Arg Ala Val Thr Val Gln Arg Leu Ala Glu Leu

180 185 190

Ala Asp Phe Tyr Gly Val Pro Val Gln Glu Leu Leu Pro Gly Thr Thr

195 200 205

Pro Gly Gly Ala Ala Glu Pro Pro Pro Lys Leu Val Leu Asp Leu Glu

210 215 220

Arg Leu Ala His Val Pro Gln Glu Lys Ala Gly Pro Leu Gln Arg Tyr

225 230 235 240

Ala Ala Thr Ile Gln Ser Gln Arg Gly Asp Tyr Asn Gly Lys Val Leu

245 250 255

Ser Ile Arg Gln Asp Asp Leu Arg Thr Leu Ala Val Ile Tyr Asp Gln

260 265 270

Ser Pro Ser Val Leu Thr Glu Gln Leu Ile Ser Trp Gly Val Leu Asp

275 280 285

Ala Asp Ala Arg Arg Ala Val Ala His Glu Glu Asn

290 295 300

<210> 25

<211> 52

<212> DNA

<213> Artificial sequence

<400> 25

ggaggcctag gcttttgcaa aaagcttgcc accatggcta gaggctgcct ca 52

<210> 26

<211> 53

<212> DNA

<213> Artificial sequence

<400> 26

gaagcggccg gccgccccga ctctagagtg gtgatggtgg tggtggtact cgg 53

<210> 27

<211> 29

<212> DNA

<213> Artificial sequence

<400> 27

ctagctagcc gagggcctat ttcccatga 29

<210> 28

<211> 28

<212> DNA

<213> Artificial sequence

<400> 28

ccggaattca taccgcacag atgcgtaa 28

<210> 29

<211> 30

<212> DNA

<213> Artificial sequence

<400> 29

ctatagaacc gatcctccca ttggcctgca 30

<210> 30

<211> 31

<212> DNA

<213> Artificial sequence

<400> 30

ggccaatggg aggatcggtt ctatagacgt t 31

<210> 31

<211> 24

<212> DNA

<213> Artificial sequence

<400> 31

caccgataga accgatcctc ccat 24

<210> 32

<211> 24

<212> DNA

<213> Artificial sequence

<400> 32

aaactttcaa tgggacaccc agcc 24

<210> 33

<211> 25

<212> DNA

<213> Artificial sequence

<400> 33

cgacgcgtac ctgacgtccg atcca 25

<210> 34

<211> 30

<212> DNA

<213> Artificial sequence

<400> 34

ccgctcgaga gagctgtttt aaaagcttta 30

<210> 35

<211> 48

<212> DNA

<213> Artificial sequence

<400> 35

tttaaactta agcttggtac gccaccatgt acccatacga tgttccag 48

<210> 36

<211> 48

<212> DNA

<213> Artificial sequence

<400> 36

ggtttaaacg ggccctctag ttagctggcc tccacctttc tcttcttc 48

<210> 37

<211> 24

<212> DNA

<213> Artificial sequence

<400> 37

caccggctgg gtgtcccatt gaaa 24

<210> 38

<211> 24

<212> DNA

<213> Artificial sequence

<400> 38

aaactttcaa tgggacaccc agcc 24

<210> 39

<211> 24

<212> DNA

<213> Artificial sequence

<400> 39

caccatggag agtttgcaag gagc 24

<210> 40

<211> 24

<212> DNA

<213> Artificial sequence

<400> 40

aaacgctcct tgcaaactct ccat 24

<210> 41

<211> 23

<212> DNA

<213> Artificial sequence

<400> 41

cacctgtact ctctgaggtg ctc 23

<210> 42

<211> 23

<212> DNA

<213> Artificial sequence

<400> 42

aaacgagcac ctcagagagt aca 23

<210> 43

<211> 23

<212> DNA

<213> Artificial sequence

<400> 43

caccgagtca ccctcctgga aac 23

<210> 44

<211> 23

<212> DNA

<213> Artificial sequence

<400> 44

aaacgtttcc aggagggtga ctc 23

<210> 45

<211> 24

<212> DNA

<213> Artificial sequence

<400> 45

caccccttgg tgaagtctcc tttg 24

<210> 46

<211> 24

<212> DNA

<213> Artificial sequence

<400> 46

aaaccaaagg agacttcacc aagg 24

<210> 47

<211> 24

<212> DNA

<213> Artificial sequence

<400> 47

caccatgtta aaatccgaaa atgc 24

<210> 48

<211> 24

<212> DNA

<213> Artificial sequence

<400> 48

aaacgcattt tcggatttta acat 24

<210> 49

<211> 23

<212> DNA

<213> Artificial sequence

<400> 49

caccacgcgt gctctccctc atc 23

<210> 50

<211> 23

<212> DNA

<213> Artificial sequence

<400> 50

aaacgatgag ggagagcacg cgt 23

<210> 51

<211> 23

<212> DNA

<213> Artificial sequence

<400> 51

caccctgtgg gttgggcctg ctg 23

<210> 52

<211> 23

<212> DNA

<213> Artificial sequence

<400> 52

aaaccagcag gcccaaccca cag 23

<210> 53

<211> 51

<212> DNA

<213> Artificial sequence

<400> 53

gatagtgctg gtagtgctgg tagtgctggt ggctccgggc gcgccgacgc g 51

<210> 54

<211> 47

<212> DNA

<213> Artificial sequence

<400> 54

gatccgagct cggtaccaag cttttagttt tcctcgtgag ccacagc 47

<210> 55

<211> 48

<212> DNA

<213> Artificial sequence

<400> 55

cttaagcttg gtaccgccac catgccttca gggcagatca gcaaccag 48

<210> 56

<211> 52

<212> DNA

<213> Artificial sequence

<400> 56

accagcacta ccagcactac cagcactatc cagtgactgg ggtggtcctg gg 52

<210> 57

<211> 37

<212> DNA

<213> Artificial sequence

<400> 57

ccggaattcg ccaccatggt gagcaagggc gaggagc 37

<210> 58

<211> 40

<212> DNA

<213> Artificial sequence

<400> 58

cccaagcttt tacttgtaca gctcgtccat gccgagagtg 40

<210> 59

<211> 52

<212> DNA

<213> Artificial sequence

<400> 59

gatccagcct ccgcggaatt cgccaccatg gcttcaaact ttactcagtt cg 52

<210> 60

<211> 53

<212> DNA

<213> Artificial sequence

<400> 60

ccagagctgt tttaaaagct ttaaaacaga gatgtgtcga agatggacag tcc 53

<210> 61

<211> 54

<212> DNA

<213> Artificial sequence

<400> 61

ccagagctgt tttaaaagct tcaatcgata tataacatat cgagatcgaa atcg 54

<210> 62

<211> 54

<212> DNA

<213> Artificial sequence

<400> 62

ccagagctgt tttaaaagct ttaggagaca gtggggtcct tggctttggg aggc 54

<210> 63

<211> 50

<212> DNA

<213> Artificial sequence

<400> 63

gtcttatact tggatcaccg aattcgccac catggcttca aactttactc 50

<210> 64

<211> 52

<212> DNA

<213> Artificial sequence

<400> 64

gtttcggtaa ggggtccgct atctagagtt gacattgatt attgactagt ta 52

<210> 65

<211> 59

<212> DNA

<213> Artificial sequence

<400> 65

atcgcgaagc agcgcaaaac gcctaaccct aagcccatag agcccaccgc atccccagc 59

<210> 66

<211> 34

<212> DNA

<213> Artificial sequence

<400> 66

gcttagggtt aggcgttttg cgctgcttcg cgat 34

<210> 67

<211> 55

<212> DNA

<213> Artificial sequence

<400> 67

cagtcgaggc tgatcagcga gctctagagg ctgatcagcg ggtttaaacg ggccc 55

Claims

1. A far-red light regulated CRISPR/Cas9 system based genome transcription apparatus, comprising: a far-red light sensing element, a far-red light effect element, and a genome-localized transcription element;

wherein the far-red photoreceptor element comprises bacterial photosensitive diguanylate cyclase BphS and a degrading enzyme YhjH of c-di-GMP;

the far-red light effect element comprises a fusion protein P65-VP64-BldD responding to c-di-GMP, and a promoter P for starting expression of a transcription activator _FRL And a transcriptional activator FGTAs that initiates transcriptional activation of a downstream gene;

the sequence of the fusion protein p65-VP64-BldD is shown in SEQ ID NO. 24; the promoter P _FRL Comprises a DNA sequence combined with BldD protein and a weak promoter for promoting gene expression, and the nucleotide sequence is selected from any one of the sequences shown in SEQ ID NO. 5-9; amino acids of the transcriptional activator FGTAsThe sequence is selected from any one of the sequences shown as SEQ ID NO. 16-19;

the genome positioning transcription element can effectively target to the site of a genome target gene promoter, and comprises dCas9 protein and single-stranded RNA with a guide targeting effect; wherein the dCas9 is a mutant D10A and H840A of the Cas9 protein, and the amino acid sequence of the mutant is shown as a sequence SEQ ID NO. 10.

2. The far-red light regulated CRISPR/Cas9 system based genome transcription device according to claim 1, wherein the amino acid sequence of BphS is as shown in SEQ ID No. 23; the coding gene sequence Genbank accession number of the degrading enzyme YhjH of the c-di-GMP is as follows: ANK04038.

3. The far-red light regulated CRISPR/Cas9 system based genome transcription device according to claim 1, wherein said weak promoter that promotes gene expression comprises tatabex, cytomegalovirus hCMV minimal promoter and its mutant hCMVmin3G promoter.

4. A kit comprising the far-red light regulated CRISPR/Cas9 system-based genome transcription apparatus of claim 1.

5. A kit comprising the eukaryotic expression vector of the far-red light regulated CRISPR/Cas9 system based genome transcription apparatus of claim 1 and/or a host cell transfected with the eukaryotic expression vector and/or an engineered cell transplantation vector and corresponding instructions.

6. Use of the far-red light regulated CRISPR/Cas9 system based genome transcription device of claim 1 in the preparation of a kit for inducing expression of an endogenous gene in a mammal.