CN110468153A

CN110468153A - A kind of subgenomic transcription device, construction method and application based on CRISPR/Cas9 system of far-red light regulation

Info

Publication number: CN110468153A
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: 2019-11-19
Anticipated expiration: 2038-05-11
Also published as: CN110468153B

Abstract

The invention discloses a kind of subgenomic transcription device based on CRISPR/Cas9 system of far-red light regulation, including far-red light primary element, far-red light response element and genome position transcriptional elements.The subgenomic transcription device based on CRISPR/Cas9 system of the far-red light regulation being capable of the endogenous transcriptional expression with foreign gene (single or multiple) of high efficiency regulatory mammal.Present system can pass through the endogenous expression with foreign gene of far-red light control accurate mammalian cell, have the characteristics that nontoxicity, high efficiency, insulating properties and Space-time speciality inducible gene expression, realizes that there is huge potential using value in the accurately research of spatiotemporal database cell behavior in mammalian genes engineering and regenerative medicine field.

Description

A kind of subgenomic transcription device based on CRISPR/Cas9 system of far-red light regulation, Construction method and application

Technical field

The present invention relates to the multi-crossed disciplines fields such as synthetic biology, light science of heredity, and in particular to a kind of far-red light regulation Subgenomic transcription device, the construction method based on CRISPR/Cas9 system, efficiently induce that mammalian cell is endogenous and external source The expression of gene and its application in mammalian genes engineering and regenerative medicine.

Background technique

In synthetic biology field, the molecular switch of artificial accuracy controlling gene expression is in mammalian genes engineering Have become a kind of indispensable means.There are many system of artificial regulatory inducible gene expression, these regulations at present System is mainly by the method for chemical inducer or physics come the expression of induction regulating controlling gene, however by photoinduction regulation The system of source gene expression is also less.

Light is a kind of ideal gene expression inducer.It is generally existing in nature, is easy to get, and has space-time special The opposite sex, and there is no toxicity.Therefore inducer is used light as to regulate and control endogenous gene expression and in the research of genetic engineering There is very much application value.

Currently, the system based on CRISPR has developed into for a kind of powerful gene editing tool, in biomedicine Field has broad application prospects.These systems are made of Cas9 nuclease and single stranded RNA (sgRNA), can be directed to specific Gene, so that it is relatively easily adjusted gene expression, including inducible gene expression, gene silencing, gene defect reparation etc..It arrives So far, the re-recording system of the light-operated activation endogenous gene of the regulation based on dCas9 is still not perfect.These are based on CRISPR/ The light-operated activated transcription system of Cas9, is substantially by blue light or ultraviolet light as inducer, but they there are certain offices It is sex-limited, such as there is certain toxicity, transdermal low efficiency is not easy to carry out the regulation of target gene through skin or abdominal cavity, this is greatly Limit deep level development and the clinical application of existing light-operated CRISPR/Cas9 system.In order to overcome these limitations, need to send out A kind of bright fast reaction, easily, efficient, safety and low toxicity the subgenomic transcription device effectively endogenous base of transcriptional activation Because of expression, for use in Study on Transformation and clinical application.

Summary of the invention

In view of the above shortcomings of the prior art, the present invention propose a kind of regulation of far-red light based on CRISPR/Cas9 system Subgenomic transcription device, inducing expression carried out to endogenous genes of mammal, the present invention has that orthogonality is good, stability The features such as high, controlling gene expresses multiple height, height Space-time speciality, strong tissue penetration and has no toxic side effect, in lactation Animal genetic engineering accurately has huge potential using value in the research of Space-time speciality regulating cell behavior.

Present invention firstly provides a kind of subgenomic transcription devices based on CRISPR/Cas9 system of far-red light regulation. In the present invention, the photon energy of the far-red light is more much lower than blue light photon energy, and the toxic side effect generated to cell is remote Much smaller than blue light.The penetrability of far-red light is far longer than blue light, can pass through skin, 7-8 centimetres of musculature, may be implemented seamless The target cell expression target gene or even controllable internal specific organization's organ expression target base of slash regulation transplanting in animal body Cause.In addition, far-red light regulator control system of the present invention is not needed additionally to add any phytochrome and can be activated by far-red light.

" CRISPR/Cas9 system " of the present invention is that finger nuclease Cas9 albumen can be with tracrRNA (trans- Activating RNA) and its complementary pairing compound combine, and corresponding sequence target site shear double-stranded DNA.dCas9 Albumen is the mutant of Cas9 albumen, do not have cleavage activity, but still can under the mediation of sgRNA accurate target gene Group DNA.

The invention also provides a kind of control systems of far-red light regulation endogenous gene expression loop.Present invention optimizes be Far-red light response element in system, the inducing effect being optimal regulate and control system to later light so that system is more sensitive to far-red light The deep level development of system and clinical application are advantageously.Far-red light controlling gene of the present invention expression loop control system can be with The expression of single or multiple endogenous genes is induced, new tool is provided for genetic engineering, there is great potential using value, It can be widely popularized in clinical application.

Artificial synthesized method preparation can be used in heretofore described each nucleotide sequence or amino acid sequence.

The invention proposes a kind of genes based on CRISPR/Cas9 system of the far-red light of engineer, synthesis regulation Group rerecording device.

The present invention also provides the eukaryon tables of the subgenomic transcription device based on CRISPR/Cas9 system of far-red light regulation Up to carrier, engineering cell.

The present invention also provides the subgenomic transcription device each groups based on CRISPR/Cas9 system of above-mentioned far-red light regulation The kit divided.

The present invention can be expressed with rapid induction endogenous gene, and can have inducing expression times with controlling gene expression quantity The features such as counting height, height Space-time speciality, strong tissue penetration and having no toxic side effect.

The subgenomic transcription device based on CRISPR/Cas9 system of far-red light regulation proposed by the present invention comprising: it is remote Feux rouges primary element, far-red light response element and genome position transcriptional elements.

In the present invention, the far-red light primary element includes the photosensitive two guanylate cyclases BphS and c-di-GMP of bacterium Degrading enzyme YhjH.The amino acid sequence of the BphS as shown in SEQ ID NO.23, the degrading enzyme YhjH's of the c-di-GMP Coding gene sequence Genebank accession number: ANK04038.

In the present invention, the far-red light response element includes the fusion protein p65-VP64-BldD for responding c-di-GMP, opens The promoter P of dynamic expression transcription activator_FRLWith the transcription activator (FGTAs) of transcriptional activation downstream gene.

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

Wherein, the promoter P_FRLThe weak starting for DNA sequence dna and the promotor gene expression for identifying and combining for BldD albumen Son composition；

The protein bound DNA sequence dna of BldD is the polypeptid specificity knowledge of DNA binding domain and c-di-GMP binding domain DNA sequence dna that is other and combining, is the partial sequence of whiG promoter region, nucleotide sequence (BldD as shown in SEQ ID NO.1 Binding site (whiG) nucleotide sequence) and whiG different copy numbers；

The weak promoter of promotor gene expression includes TATAbox, nucleotide sequence as shown in SEQ ID NO.2, it is huge Cell virus hCMV minimal promoter, nucleotide sequence is as shown in SEQ ID NO.3 and its mutant hCMVmin 3G, nucleosides Acid sequence is as shown in SEQ ID NO.4.

The weak starting for DNA sequence dna and the promotor gene expression for identifying and combining present invention optimizes the BldD in effector The weak promoter of son, DNA sequence dna and the promotor gene expression that the BldD is identified and combined can be selected from nucleotide sequence such as SEQ P shown in ID NO.5_FRL1a:pA-3*whiG-P_hCMVmin, the P as shown in SEQ ID NO.6_FRL2a:3*whiG-P_hCMVmin, such as SEQ P shown in ID NO.7_FRL3a:1*whiG-P_hCMVmin, the P as shown in SEQ ID NO.8_FRL1b:2*whiG-P_hCMVmin3G, such as SEQ P shown in ID NO.9_FRL2b:3*whiG-P_hCMVmin3G。

Wherein, the albumen of transcription activator (FGTAs) coding of the far-red light induction can activate endogenous gene Transcription activator, the various combination including VP64, p65, MS2, VPR and HSF1.Wherein, the VP64 is herpes simplex virus The transcriptional activation domain of granule protein VP16 transcriptional activation domain and its 4 copy, amino acid sequence such as SEQ ID Shown in NO.11.Wherein, the p65 is NF-KB p65 subunit transcriptional activation domain, amino acid sequence such as SEQ ID Shown in NO.12.Wherein, the MS2 is a kind of coat protein, and amino acid sequence is as shown in SEQ ID NO.13.Wherein, described VPR is the fusion protein of VP64-p65-Rta, and amino acid sequence is as shown in SEQ ID NO.14.Wherein, the HSF1 is heat Shock transcription factor HSF1 transcription activating domain, amino acid sequence is as shown in SEQ ID NO.15.Wherein, the various combination ammonia The optional SEQ of the sequence freely ID NO.16-19 of base acid sequence, including FGTA1:MS2-Linker-NLS-VP64, FGTA2:MS2- Linker-NLS-VPR、FGTA3:MS2-Linker-NLS-p65-HSF1-VP64、FGTA4:MS2-Linker-NLS-p65- HSF1 etc..Wherein, when the transcription activator of far-red light induction is FGTA4:MS2-Linker-NLS-p65-HSF1, promoter is P_FRL1bWhen induced activation efficiency highest.

In the present invention, genome positioning transcriptional elements include dCas9 albumen and with instructing the single-stranded of targeting RNA(sgRNA)。

Wherein, the dCas9 is the mutant (D10A, H840A) of Cas9 albumen, amino acid sequence such as SEQ ID Shown in NO.10；The single stranded RNA includes the gRNA that can target target gene promoter.

The mechanism of action of the subgenomic transcription device based on CRISPR/Cas9 system of far-red light of the present invention regulation is, when When generating c-di-GMP under far-red light is according to irradiation condition, c-di-GMP forms it into dimer in conjunction with BldD, activates downstream Access expresses transcription activator, then by conjunction with MS2 loop-stem structure, activated gene transcriptional expression.When illumination stopping cannot When generating c-di-GMP, the c-di-GMP synthesized is degraded, and BldD can not form dimer, then can not start transcription activator Expression, target gene can not be transcribed.

The gene based on CRISPR/Cas9 system that can be regulated and controled far-red light provided by the invention by technique for gene engineering Three component constructions of group rerecording device realize the transcriptional expression of regulation target gene in carrier for expression of eukaryon.This hair The subgenomic transcription device based on CRISPR/Cas9 system of the far-red light regulation of bright offer is thin using hardly damage Expression of the specificity induction endogenous gene in eukaryotic host cell over time and space of the far-red light of born of the same parents or body, the place Chief cell can be any type of mammalian cell, such as hMSC-TERT, Hana 3A, HEK-293A, HEK-293T etc..

Wherein, the intensity of illumination of the far-red light is 0-5mW/cm²；Irradiation time is 0-72h；Illuminating method includes pulse Formula irradiation, Continuous irradiation or direct irradiation spatially control the gene expression dose of the cell of different location.It is remote by controlling Red-light source generates different light application times, to realize the different degrees of induction to endogenous gene.The far-red light light source The device of 600-900nm wavelength far-red light can be generated, can be 600-900nm LED, infrared therapeutic device, laser lamp etc..

Present invention optimizes far-red light response element, the promoter P including starting expression transcription activator_FRLSwash with transcription The transcription activator (FGTAs) of downstream gene living.(1) present invention optimizes promoter P_FRL, wherein the promoter P_FRLInclude The weak promoter sequence that the BldD of dimerization identify under c-di-GMP effect and the DNA sequence dna and promotor gene that combine are expressed. The weak promoter of the promotor gene expression can be arbitrary weak promoter, such as nucleotide sequence such as sequence SEQ ID NO.2 Shown in TATA box, such as nucleotide sequence cytomegalovirus minimal promoter hCMV as shown in sequence SEQ ID NO.3_min, And its mutant hCMVmin 3G etc., nucleotide sequence is as shown in SEQ ID NO.4.The DNA sequence that the BldD is identified and combined Column and promoter can be selected from nucleotide sequence P as shown in SEQ ID NO.5_FRL1a:pA-3*whiG-P_hCMVmin, such as SEQ ID P shown in NO.6_FRL2a:3*whiG-P_hCMVmin, the P as shown in SEQ ID NO.7_FRL3a:1*whiG-P_hCMVmin, such as SEQ ID P shown in NO.8_FRL1b:2*whiG-P_hCMVmin3G, the P as shown in SEQ ID NO.9_FRL2b:3*whiG-P_hCMVmin3G, can make The system induction multiple is higher.(2) present invention optimizes transcription activator, the transcription activator 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 etc., amino acid sequence is as shown in SEQ ID NO.16,17,18,19.

The invention also provides the structures of the subgenomic transcription device based on CRISPR/Cas9 system of far-red light regulation Construction method, comprising the following steps:

(1) far-red light primary element is constructed

Building is as the polypeptide of DNA binding domain and c-di-GMP binding domain, the polypeptide as nuclear localization signal NLS, conduct Far-red light primary element of the complex of the polypeptide of link field and the polypeptide as transcriptional regulatory domain as system.

Wherein, the polypeptide as DNA binding domain and c-di-GMP binding domain, after in conjunction with c-di-GMP, energy Albumen in conjunction with specific DNA sequence dna, including BldD albumen, amino acid sequence is as shown in SEQ ID NO.20；

Wherein, the polypeptide as nuclear localization signal NLS can copy diversified forms, amino acid sequence for 1-3 As shown in SEQ ID NO.21；

Wherein, the polypeptide as linkage function domain, length can be from 0-30 amino acid diversified forms, amino Acid sequence is as shown in SEQ ID NO.22；

Wherein, the polypeptide as transcriptional regulatory domain is the domain protein with transcriptional activation function.

Wherein, the polypeptide as transcriptional regulatory domain is placed in the polypeptide of the DNA binding domain and c-di-GMP binding domain The N-terminal or C-terminal of BldD.

(2) far-red light response element is constructed.

Wherein, far-red light response element includes promoter P_FRLWith transcription activator (FGTAs), it is expressed as P_FRL-FGTAs。 The promoter P_FRLIt can also be formed for the protein bound DNA sequence dna of BldD and weak promoter, wherein the processor BldD egg The DNA sequence dna of white combination, for the polypeptid specificity DNA sequence dna that identifies and combine of DNA binding domain and c-di-GMP binding domain, For the partial sequence of whiG promoter region, the copy form that nucleotide sequence is as shown in SEQ ID NO.1 and its is different； The weak promoter of the promotor gene expression includes all weak promoters comprising TATAbox, cytomegalovirus CMV are minimum Promoter and its mutant CMVmin 3G.

(3) building genome positions transcriptional elements.

Wherein, genome positioning transcriptional elements include dCas9 albumen and with the single stranded RNA for instructing targeting (sgRNA)；

Wherein, the dCas9 is the mutant D10A, H840A, amino acid sequence such as sequence SEQ ID of Cas9 albumen Shown in NO.10.

Wherein, have and instruct the single stranded RNA (sgRNA) of targeting as matching target gene promoter can be combined, and In conjunction with the arbitrary RNA sequence of dCas9 albumen.

The invention also provides a kind of kit, containing far-red light regulation based on CRISPR/Cas9 system Subgenomic transcription device.The invention also provides a kind of kits, containing far-red light regulation based on CRISPR/Cas9 The carrier for expression of eukaryon of the subgenomic transcription device of system and/or transfected the carrier for expression of eukaryon host cell and/or It is engineered cell transplantation carrier and corresponding specification.

In the present invention, the kit includes regulating and controlling the far-red light controlling gene to express loop control system each group sub-prime Grain kit, containing the mammalian cell kit for regulating and controlling far-red light controlling gene expression loop control system and Corresponding specification.

The subgenomic transcription based on CRISPR/Cas9 system the invention also provides preparation containing far-red light regulation Carrier for expression of eukaryon, engineering cell or the method for being engineered cell transplantation carrier of device.

The carrier for expression of eukaryon includes that the genome based on CRISPR/Cas9 system containing far-red light regulation turns The mammalian cell expression vector of recording device.The expression vector, which can be, individually encodes base containing far-red light primary element The carrier of cause or carrier individually containing far-red light response element encoding gene are individually compiled containing genome positioning transcriptional elements The carrier of code gene, the far-red light response element include the promoter (P of far-red light response_FRL) and transcription activator (FGTA).Alternatively, the expression vector includes the carrier of far-red light primary element encoding gene, far-red light response element coding base Two or three in the carrier of cause, the carrier of genome positioning transcriptional elements encoding gene.Aforementioned all mammals See Table 1 for details for the building mode of fibrocyte expression vector.

The invention also provides the subgenomic transcription devices based on CRISPR/Cas9 system containing far-red light regulation Carrier for expression of eukaryon induction endogenous genes of mammal expression in application.

The beneficial effects of the present invention are: the gene based on CRISPR/Cas9 system of far-red light regulation of the present invention Group rerecording device can induce endogenous gene expression quickly through far-red light, and control accurate gene expression has induced gene table The features such as up to multiple height, height Space-time speciality, strong tissue penetration and having no toxic side effect.

Detailed description of the invention

Fig. 1 is the subgenomic transcription principle of device schematic diagram based on CRISPR/Cas9 system of far-red light regulation；

Fig. 2 is the subgenomic transcription device based on CRISPR/Cas9 system that different transcription activators regulate and control far-red light Activation efficiency result figure；

Fig. 3 is that different promoters induce the subgenomic transcription device based on CRISPR/Cas9 system that far-red light regulates and controls Activation efficiency result figure；

Fig. 4 is that different illumination intensity lures the subgenomic transcription device based on CRISPR/Cas9 system that far-red light regulates and controls The activation efficiency result figure led；

Fig. 5 is the subgenomic transcription device based on CRISPR/Cas9 system that different light application times regulate and control far-red light Activation efficiency result figure；

Fig. 6 be the subgenomic transcription device based on CRISPR/Cas9 system of far-red light regulation in different cell lines Activation efficiency result figure；

Fig. 7 is the reversible control verifying of the subgenomic transcription device based on CRISPR/Cas9 system of far-red light regulation；

Fig. 8 is the Region-specificity verifying of the subgenomic transcription device based on CRISPR/Cas9 system of far-red light regulation；

Fig. 9 is the subgenomic transcription device induction based on CRISPR/Cas9 system that different gRNA regulate and control far-red light Activate endogenous gene efficiencies figure；

Figure 10 is that different illumination intensity lures the subgenomic transcription device based on CRISPR/Cas9 system that far-red light regulates and controls The activation endogenous gene efficiencies figure led；

Figure 11 is the subgenomic transcription device based on CRISPR/Cas9 system that different light application times regulate and control far-red light Activate endogenous gene efficiencies figure；

Figure 12 be the subgenomic transcription device based on CRISPR/Cas9 system of far-red light regulation in different cell lines Activate endogenous gene efficiencies figure；

Figure 13 be the subgenomic transcription device based on CRISPR/Cas9 system of far-red light regulation activate endogenous gene can Inverse control is verified；

Figure 14 is the subgenomic transcription device based on CRISPR/Cas9 system of far-red light regulation while activating multiple endogenous Gene efficiencies figure.

Specific embodiment

In conjunction with following specific embodiments and attached drawing, the present invention is described in further detail.These embodiments are only used for Invention is illustrated, without constituting any restrictions to the scope of the present invention.Implement procedures, conditions, experimental methods etc. of the invention, In addition to what is specifically mentioned below, it is among the general principles and common general knowledge in the art, the present invention is not particularly limited interior Hold.Reagent used in following embodiment, instrument etc. and test method without specific conditions, according to routine or commodity Condition proposed by supplier carries out.

Materials and methods

Molecular cloning

All expression plasmids of the molecule clone technology building present invention, step is common sense in the industry.

All primers for PCR are synthesized by Jin Weizhi Biotechnology Co., Ltd.It is constructed in the embodiment of the present invention Expression plasmid all passes through sequencing, and sequencing is completed by Jin Weizhi Biotechnology Co., Ltd.Institute in the embodiment of the present invention Phanta Max Super-Fidelity archaeal dna polymerase is purchased from Nanjing Vazyme Biotechnology Co., Ltd..In nucleic acid Enzyme cutting, T4DNA ligase are purchased from TaKaRa company.Homologous recombination enzyme is purchased from and the limited public affairs of first biotechnology (Shanghai) share Department.Phanta Max Super-Fidelity archaeal dna polymerase is accompanied with corresponding polymerase buffer and dNTP when buying.Core Corresponding buffer is accompanied with when sour restriction endonuclease, T4 DNA ligase, the purchase of homologous recombination enzyme.Yeast extract (Yeast Extract), tryptone (Trypton), agar powder, ammonia benzyl blueness enzyme plain (Amp) are limited purchased from the raw work biotechnology in Shanghai Company.DNA Marker DL5000, DNAMarker DL2000 (precious bioengineering Co., Ltd)；Nucleic acid dye EB (Guangdong state Biotech company difficult to understand)；The small pumping extracts kit (TIANGEN Biotech (Beijing) Co., Ltd.) of plasmid；DNA glue reclaim reagent It is century Biotechnology Co., Ltd that box, PCR product purification kit, which are purchased from health,；The dehydrated alcohol that is referred in embodiment, Remaining reagent such as NaCl is domestic analysis net product.The glue recycling of DNA fragmentation, purification and recovery, step are recycled according to DNA glue The operational manual of kit, PCR product purification kit (health is century Biotechnology Co., Ltd)；Plasmid extraction step root According to small pumping (TIANGEN Biotech (Beijing) Co., Ltd.) the extracts kit specification of plasmid.

Cell culture and transfection

In the embodiment of the present invention with following cell line and PEI transfection for illustrate far-red light regulation based on CRISPR/ Working condition of the subgenomic transcription device of Cas9 system in cell, but do not limit the scope of the present invention.

Centrifuge tube for the 10cm Tissue Culture Dish of cell culture, tissue culture plate (24 hole), 15mL and 50mL is purchased From Thermo Fisher Scientific company, the U.S. (Labserv)；Improved Eagle culture medium, the tire ox blood used Clearly, penicillin and Streptomycin Solution are purchased from U.S. Gibico company；Transfection PEI used is purchased from Polysciences company；Carefully Born of the same parents' incubator is purchased from U.S. Thermo Fisher Scientific company；Remaining consumptive material is common domestic consumptive material.

Cell culture: the cell being related in patent includes human embryonic kidney cell (HEK-293, ATCC:CRL-11268), One E1 gene (ThermoFisher, R70507) copy of stable integration, the human mesenchymal stem cell (hmsct- of telomere Tert26 it) is trained in improved Eagle culture medium (DMEM) with Hana3A cell derived from HEK-293, all cell culture Support the penicillin and Streptomycin Solution of fetal calf serum and 1% (v/v) that 10% (v/v) is added in base；Cell culture in 37 DEG C, contain In the incubator for having 5% gas concentration lwevel.

Transfection: all cell line transfections using the PEI after optimization operating procedure (Wieland M, Methods 56 (3): 351).Briefly, it is as inoculated in the 10cm Tissue Culture Dish that cultivating system is 10mL, × 10⁴A cell is being cultivated After 18h, the DNA of best proportion is stood into 6h according to the mass ratio and PEI mixed dissolution of 3:1 (PEI:DNA) in culture medium. (polyethyleneimine, molecular weight 40,000, stoste 1mg/mL in ddH₂O；Polysciences；Cat.no.24765) cell number Meter number is counted with Countess II automated cell counter.

The detection of reporter gene secreted alkaline phosphatase (SEAP)

Homoarginine, magnesium chloride, diethanol amine, HCl for configuring examining report Gene response buffer are purchased to life Work bioengineering (Shanghai) limited liability company；Chromogenic substrate (p-nitrophenylphosphate) is purchased to the brilliant pure biochemical technology stock in Shanghai Part Co., Ltd (Aladdin).

(1) reagent configures:

2x buffer:

20mM homoarginine note: it is to inhibit endogenic alkaline phosphatase activities that it, which is acted on,

1mM magnesium chloride

2% diethanol amine

PH to 9.8 is adjusted with HCl

Substrate solution:

120mM chromogenic substrate (p-nitrophenylphosphate)

2x measures buffer

(2) experimental procedure:

1. drawing cell culture supernatant liquid, into centrifuge tube, (note: generally will be more than 150 μ L to 200 μ L, because subsequent add Volume a part can be lost in heat).

(note: heating mainly allows endogenic alkaline phosphatase to inactivate to 2.65 DEG C of water-bath 30min, and SEAP high temperature resistant, In It will not be inactivated at a temperature of this).

3. drawing 80 μ L (experimental situation voluntarily dilutes) to 96 orifice plates, rapidly join preheated in advance 20 μ l of 100 μ l of 2xbuffer and substrate solution.

4. microplate reader 405nm is surveyed 10 times, every minor tick 1min (experimental situation can separately set condition).

(3) calculating of enzyme activity

The enzyme activity definition of alkaline phosphatase (SEAP) is: 37 DEG C, when pH 9.8, in 1min with substrate p-nitrophenyl phosphorus Acid disodium (PNPP-Na₂) alkaline phosphatase for generating 1mol/L p-nitrophenol is reacted, it is defined as 1 unit of activity (1U).It is right Nitrophenol itself has glassy yellow, and in wavelength 405nm, the p-nitrophenol of various concentration corresponds to different light absorption values.It calculates Method are as follows: it is enzyme activity that the surveyed OD value of different time points, which is made into slope of a curve * 256.8, during sample and substrate reactions, single Position U/L.

Gene activation in the mammalian cell of far-red light control

Endogenous Gene Activation experiment.6 × 10 are inoculated in the 10cm Tissue Culture Dish that cultivating system is 10mL⁴A cell, Cultivate 18h.Transfect cell with the PEI:DNA of the 3:1 of 50 μ l mixed systems, comprising 0.31 μ g (FACE-v1.x total DNA amount) or The DNA of 0.375 μ g (FACE-v2.x total DNA amount).After transfection for 24 hours, with the far-red light LED of the 4*6 array of customization (730nm, Epistar, Taiwan, China) with different light application times (0-6h), intensity of illumination (0-2mW/cm²) carry out illumination.Transfection 48h afterwards measures SEAP value.HEK-293 cell pWS46, pGY32, pGY102, pSZ69 (each 100ng) and sgRNAs (50ng) Cotransfection, then daily illumination 6h ((1.5mW/cm²；730nm)).Total serum IgE is extracted after first time illumination 48h, is carried out real-time Quantitative fluorescent PCR.

The Region-specificity of gene activation in the mammalian cell of far-red light control

3 × 10 are planted in the culture dish of diameter 10cm⁶A HEK-293 cell after cultivating 18h, transfects 1500 μ l mixtures The PEI:DNA of the 3:1 of system transfects cell, and comprising 9.3 μ g (FACE-v1.x total DNA amount) or 11.25 μ g, (FACE-v2.x is total Amount of DNA) DNA.After transfecting 18h, pass through light shield far-red light (730nm, the 1.5mW/cm of design²) illumination 6h.After illumination 48h With Clinx imaging equipment (ChemiScope 4300Pro, Clinx, Shanghai, China) measurement fluorescence letter Number.

Quantitative RT PCR analysis

According to kit step RNAiso Plus kit (Takara, Dalian, China；Cat.no.9108 it) collects Cell extraction total serum IgE.According to step PrimeScript RT reagent Kit with the gDNA Eraser (Takara,Dalian,China；Cat.no.RR047) 1 μ g RNA reverse transcription at cDNA.Pass through Real-Time PCR Instrument (QuantStudio 3, Thermo Fisher Scientific Inc., Waltham, MA, USA) uses SYBR Premix Ex Taq(Takara,Dalian,China；Cat.no.RR420 target gene) is measured, qPCR analysis is carried out.PCR expands Increasing condition be first 95 DEG C initial denaturation ten minutes, then (95 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C extend 40 circulations 30s), last 72 DEG C of extensions 10min. the primer is shown in Table one.All samples are all with housekeeping gene glyceraldehyde 3- phosphate dehydrogenase (GAPDH) it is used as internal reference, is as a result expressed as with the opposite rna level under dark condition for reference.

Case study on implementation 1

Using SEAP as reporter gene, the genome based on CRISPR/Cas9 system of verifying far-red light regulation turns this example Recording device difference transcription activator but does not limit protection scope of the present invention the activation efficiency of gene.Specific step It is rapid as follows:

The first step, plasmid construction.See Table 1 for details for plasmid construction in this example.

Second step, inoculating cell.It will be planted after the 0.25% pancreatin digestion of the good HEK-293T cell of growth conditions In 2 piece of 24 orifice plate, every hole kind 6 × 10⁴A cell, and DMEM culture medium of the 500 μ L containing 10%FBS is added.

Third step, transfection.2 piece of 24 orifice plate is divided into dark group and light group, every group is divided into 4 groups.In inoculating cell 16 to interior for 24 hours, the addition pWS46 (P in 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 different transcriptional activations Sub- FGTA1 (pGY48) or FGTA2 (pGY51) or FGTA3 (pGY54) or FGTA4 (pGY57), with 2:2:1:1:1:2 (w/w/w/ W/w the DMEM of ratio and PEI transfection reagent and serum-free) is mixed, and is dropped evenly after being stored at room temperature 15min to 24 well culture plates In.Wherein the preparation total volume in every hole is 50 μ L, and plasmid and PEI mass ratio are 1:3.500 μ L are changed to after transfection 6h containing 10%FBS DMEM culture medium cultivated.

4th step, illumination.After changing liquid 14-18h, it is 730nm that light group, which is placed in wavelength, distinguishes 1.5mW/ in intensity of illumination cm²LED under illumination 6h, and dark group is then placed in always culture at dark.

5th step, examining report gene.

The results show that in the subgenomic transcription device based on CRISPR/Cas9 system of far-red light regulation, when activation is When FGTA4, activation efficiency is the most significant.Experimental data is detailed in Figure of description 2, and all data are all with the independently duplicated reality of n=3 The mode tested is presented.

Case study on implementation 2

Using SEAP as reporter gene, the genome based on CRISPR/Cas9 system of verifying far-red light regulation turns this example Different promoters but do not limit protection scope of the present invention the activation efficiency of gene in recording device.Specific steps It is as follows:

Second step, inoculating cell.(the specific steps are the same as those in embodiment 1)

Third step, transfection.2 piece of 24 orifice plate is divided into dark group and light group, every group is divided into 5 groups.It is thin in inoculation Born of the same parents 16 arrive in for 24 hours, and pWS46 (100ng) is added in each group, pGY32, pWS137, pWS107, pSZ69 and different promoters P_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] expression transcription swash Sub- FGTA4 (MS2-p65-HSF1) living, with the ratio of 4:4:2:2:2:1 (w/w/w/w/w) and PEI transfection reagent and serum-free DMEM is mixed, and is dropped evenly after being stored at room temperature 15min into 24 well culture plates.Wherein the preparation total volume in every hole is 50 μ L, matter Grain is 1:3 with PEI mass ratio.DMEM culture medium of the 500 μ L containing 10%FBS is changed to after transfection 6h to be cultivated.

4th step, illumination.(the specific steps are the same as those in embodiment 1)

5th step, examining report gene.

The results show that in the subgenomic transcription device based on CRISPR/Cas9 system of far-red light regulation, when promoter is P_FRL1b[(whiG)₂-P_hCMVmin3G；PGY102] when, activation efficiency is most significant.Experimental data is detailed in Figure of description 3, all Data are all presented in a manner of the independently duplicated experiment of n=3.

Case study on implementation 3

Using SEAP as reporter gene, the genome based on CRISPR/Cas9 system of verifying far-red light regulation turns this example The activation efficiency of recording device different illumination intensity, but protection scope of the present invention is not limited.Specific step is as follows:

Second step, inoculating cell.Will the good HEK-293 cell of growth conditions with 0.25% pancreatin digestion after kind in In 7 piece of 24 orifice plate, every hole kind 6 × 10⁴A cell, and DMEM culture medium of the 500 μ L containing 10%FBS is added.

Third step, transfection.It is divided into 7 groups.It is arrived in for 24 hours in inoculating cell 16, pWS46, pGY32 is added in each group, PGY102, pSZ69, pWS137 and pWS107 with the ratio of 4:4:1:2:2:2 (w/w/w/w/w/w) and PEI transfection reagent and The DMEM of serum-free is mixed, and is dropped evenly after being stored at room temperature 15min into 24 well culture plates.Wherein the preparation total volume in every hole is 50 μ L, plasmid and PEI mass ratio are 1:3.DMEM culture medium of the 500 μ L containing 10%FBS is changed to after transfection 6h to be cultivated.

4th step, illumination.After changing liquid 14-18h, placing it in wavelength is 730nm, intensity of illumination be respectively 0,0.025, 0.05、0.75、1、1.5、2mW/cm²LED under illumination 4h, illumination, which terminates to be placed in dark, cultivates.

5th step, examining report gene.

The results show that far-red light regulation the subgenomic transcription device based on CRISPR/Cas9 system in, intensity of illumination with Activation efficiency is positively correlated, intensity of illumination 1mW/cm²Activation efficiency is slowly close to saturation when above.Experimental data is detailed in explanation Book attached drawing 4, all data are all presented in a manner of the independently duplicated experiment of n=3.

Case study on implementation 4

This example using SEAP as reporter gene, verify different light application times to far-red light regulate and control based on CRISPR/Cas9 The activation efficiency of the subgenomic transcription device of system, but protection scope of the present invention is not limited.Specific step is as follows:

4th step, illumination.After changing liquid 14-18h, placing it in wavelength is 730nm, intensity of illumination 1.5mW/cm²LED Under (specific connection type referring to materials and methods) illumination 0,0.25,0.5,1,2,4,6h respectively, be immediately placed on after illumination It is cultivated at dark.

5th step, examining report gene.

The results show that in the subgenomic transcription device version 2 .2 based on CRISPR/Cas9 system of far-red light regulation, light It is positively correlated according to time and activation efficiency, activation efficiency has maximum activation efficiency in illumination 6 hours slowly close to saturation after 4 hours. Experimental data is detailed in Figure of description 5, and all data are all presented in a manner of the independently duplicated experiment of n=3.

Case study on implementation 5

Using SEAP as reporter gene, the genome based on CRISPR/Cas9 system of verifying far-red light regulation turns this example The activation efficiency of recording device in different cell lines, but protection scope of the present invention is not limited.Specific step is as follows:

Second step, inoculating cell.By the good HeLa cell of growth conditions, HEK-293A cell, Hana3A cell, Kind is in 2 piece of 24 orifice plate after Hmsc-TERT cell, the 0.25% pancreatin digestion of HEK-293 cell, every hole kind 6 × 10⁴It is a thin Born of the same parents, and DMEM culture medium of the 500 μ L containing 10%FBS is added.

Third step, transfection.2 piece of 24 orifice plate is divided into dark group and light group, every group be divided into different cell lines it is 5 small Group.It is arrived in for 24 hours in inoculating cell 16, pWS46, pGY32, pGY102, pSZ69, pWS137 and pWS107 is added in each group, with The ratio and PEI transfection reagent of 4:4:1:2:2:2 (w/w/w/w/w) and the DMEM of serum-free are mixed, and are stored at room temperature after 15min It is even to be added drop-wise in 24 well culture plates.Wherein the preparation total volume in every hole is 50 μ L, and plasmid and PEI mass ratio are 1:3.After transfecting 6h DMEM culture medium of the 500 μ L containing 10%FBS is changed to be cultivated.

5th step, examining report gene.

The results show that in the subgenomic transcription device version 2 .2 based on CRISPR/Cas9 system of far-red light regulation, In HEK-293 cell has optimal activation efficiency.Experimental data is detailed in Figure of description 6, and all data are all with n=3 independence The mode of duplication experiment is presented.

Case study on implementation 6

Using SEAP as reporter gene, the genome based on CRISPR/Cas9 system of verifying far-red light regulation turns this example The invertibity of recording device activated gene, but protection scope of the present invention is not limited.Specific step is as follows:

Third step, transfection.2 piece of 24 orifice plate is divided into two groups.It arrives in for 24 hours in inoculating cell 16, is added in each group PWS46, pGY32, pGY102, pSZ69, pWS137 and pWS107 with the ratio of 4:4:1:2:2:2 (w/w/w/w/w/w) with The DMEM of PEI transfection reagent and serum-free is mixed, and is dropped evenly after being stored at room temperature 15min into 24 well culture plates.Wherein every hole Preparation total volume be 50 μ L, plasmid and PEI mass ratio are 1:3.DMEM culture of the 500 μ L containing 10%FBS is changed to after transfection 6h Base is cultivated.

4th step, illumination.After changing liquid 14-18h, being placed in wavelength for first group is 730nm, intensity of illumination 1.5mW/cm² LED under (specific connection type referring to materials and methods) illumination 20min, culture at dark is immediately placed on after illumination, the Three days continuation illumination 20min, second group is first placed at dark and cultivates, second day illumination 20min, be placed on culture at dark.

5th step, every 6h examining report gene are every to replace culture medium for 24 hours.

The results show that far-red light regulation the subgenomic transcription device based on CRISPR/Cas9 system have it is good can Inverse modulability.Experimental data is detailed in Figure of description 7, and all data are all presented in a manner of the independently duplicated experiment of n=3.

Case study on implementation 7

This example is using GFP as reporter gene, the subgenomic transcription based on CRISPR/Cas9 system of verifying far-red light regulation The Region-specificity for filling inducible gene expression, as shown in 8 left side of attached drawing, but does not limit protection scope of the present invention.Specifically Steps are as follows:

Second step, inoculating cell.Will the good HEK-293 cell of growth conditions with 0.25% pancreatin digestion after kind in In two culture dishes, it is inoculated with 3.5 × 10 respectively⁶Cell, and DMEM culture medium of the 15mL containing 10%FBS is added.

Third step, transfection.Culture dish is divided into two groups, light group and dark group are separately added into pWS46 in each group, PSTING, pSZ70, pSZ69, pWS137 and pGY47 (P_FACE- EGFP-pA) with the ratio of 10:1:5:5:5:5 (w/w/w/w/w) It mixes with the DMEM of PEI transfection reagent and serum-free, is dropped evenly in culture dish after being stored at room temperature 15min, plasmid and PEI matter Amount is than being 1:3.Change DMEM culture medium of the 15mL containing 10%FBS is cultivated after transfection 6h.

5th step, test experience result.Cell in fluorescence microscopy microscopic observation culture dish, and take pictures.

The results show that the subgenomic transcription device energy Region-specificity based on CRISPR/Cas9 system of far-red light regulation Ground inducible gene expression.Experimental data is detailed in Figure of description 8, and all data are all in a manner of the independently duplicated experiment of n=3 It is existing.

Case study on implementation 8

Using ASCL1 as reporter gene, the genome based on CRISPR/Cas9 system of verifying far-red light regulation turns this example Influence of the gRNA to activation efficiency in recording device, but protection scope of the present invention is not limited.Specific step is as follows:

Second step, inoculating cell.By kind after the 0.25% pancreatin digestion of the good HEK-293 cell of growth conditions in 2 In 24 orifice plate of block, every hole kind 6 × 10⁴A cell, and DMEM culture medium of the 500 μ L containing 10%FBS is added.

2 piece of 24 orifice plate is divided into dark group and light group by third step, transfection, and every group is divided into 4 groups.It is arrived in inoculating cell 16 In for 24 hours, pWS46, pGY32, pGY102, pSZ69 is added in the first set respectively, pWS46, pGY32 are added in the second set, pGY102,pSZ69,pSZ83(P_U6- gRNA1 (ASCL1)-pA), pWS46, pGY32, pGY102 is added in third group, pSZ69,pSZ84(P_U6- gRNA2 (ASCL1)-pA), pWS46, pGY32, pGY102, pSZ69, pSZ83 is added in the 4th group (P_U6-gRNA1(ASCL1)-pA),pSZ84(P_U6- gRNA2 (ASCL1)-pA), it is mixed with the DMEM of PEI transfection reagent and serum-free It is even, it drops evenly after being stored at room temperature 15min into 24 well culture plates.Wherein the preparation total volume in every hole is 50 μ L, plasmid and PEI Mass ratio is 1:3.DMEM culture medium of the 500 μ L containing 10%FBS is changed to after transfection 6h to be cultivated.

5th step, examining report gene.Real-time fluorescence quantitative PCR.

The results show that adding in the subgenomic transcription device version 2 .2 based on CRISPR/Cas9 system of far-red light regulation Enter two gRNA with better activation efficiency.Experimental data is detailed in Figure of description 9, and all data are all independently multiple with n=3 The mode of system experiment is presented.

Case study on implementation 9

Using ASCL1 as reporter gene, the genome based on CRISPR/Cas9 system of verifying far-red light regulation turns this example The activation efficiency of recording device different illumination intensity, but protection scope of the present invention is not limited.Specific step is as follows:

Third step, transfection.It is divided into 7 groups.It is arrived in for 24 hours in inoculating cell 16, pWS46, pGY32 is added in each group, PGY102, pSZ69, pSZ83 and pSZ84 are with the ratio of 4:4:1:2:2:2 (w/w/w/w/w/w) and PEI transfection reagent and without blood Clear DMEM is mixed, and is dropped evenly after being stored at room temperature 15min into 24 well culture plates.Wherein the preparation total volume in every hole is 50 μ L, plasmid and PEI mass ratio are 1:3.DMEM culture medium of the 500 μ L containing 10%FBS is changed to after transfection 6h to be cultivated.

4th step, illumination.After changing liquid 14-18h, placing it in wavelength is 730nm, intensity of illumination be respectively 0,0.025, 0.05、0.75、1、1.5、2mW/cm²LED under illumination 4h, illumination terminate to be placed at dark cultivate.

5th step, examining report gene.Real-time fluorescence quantitative PCR.

The results show that far-red light regulation the subgenomic transcription device based on CRISPR/Cas9 system in, intensity of illumination with Activation efficiency is positively correlated, intensity of illumination 2mW/cm²When activation efficiency it is most significant.Experimental data is detailed in Figure of description 10, All data are all presented in a manner of the independently duplicated experiment of n=3.

Case study on implementation 10

This example using ASCL1 as reporter gene, verify different light application times to far-red light regulate and control based on CRISPR/Cas9 The activation efficiency of the subgenomic transcription device of system, but protection scope of the present invention is not limited.Specific step is as follows:

4th step, illumination.After changing liquid 14-18h, placing it in wavelength is 730nm, intensity of illumination 1.5mW/cm²LED Lower illumination 0,0.25,0.5,1,2,4,6h respectively is immediately placed at dark after illumination and cultivates.

5th step, examining report gene.

The results show that far-red light regulation the subgenomic transcription device based on CRISPR/Cas9 system, light application time and swash Active rate is positively correlated, and activation effect is the most significant when illumination 6h.Experimental data is detailed in Figure of description 11, and all data are all It is presented in a manner of the independently duplicated experiment of n=3.

Case study on implementation 11

Using ASCL1 as reporter gene, the genome based on CRISPR/Cas9 system of verifying far-red light regulation turns this example The activation efficiency of recording device in different cell lines, but protection scope of the present invention is not limited.Specific step is as follows:

Third step, transfection.2 piece of 24 orifice plate is divided into dark group and light group, every group be divided into different cell lines it is 5 small Group.It is arrived in for 24 hours in inoculating cell 16, pWS46, pGY32, pGY102, pSZ69, pSZ83 and pSZ84 is added in each group, with 4: The ratio and PEI transfection reagent of 4:1:2:2:2 (w/w/w/w/w) and the DMEM of serum-free are mixed, and are stored at room temperature after 15min uniformly It is added drop-wise in 24 well culture plates.Wherein the preparation total volume in every hole is 50 μ L, and plasmid and PEI mass ratio are 1:3.It is changed after transfection 6h Enter DMEM culture medium of the 500 μ L containing 10%FBS to be cultivated.

5th step, examining report gene.

The results show that the subgenomic transcription device based on CRISPR/Cas9 system of far-red light regulation, thin in HEK-293 Born of the same parents have optimal activation efficiency.Experimental data is detailed in Figure of description 12, and all data are all with the independently duplicated experiment of n=3 Mode present.

Case study on implementation 12

Using ASCL1 as reporter gene, the genome based on CRISPR/Cas9 system of verifying far-red light regulation turns this example The invertibity of recording device activated gene, but protection scope of the present invention is not limited.Specific step is as follows:

Second step, inoculating cell.Will the good HEK-293 cell of growth conditions with 0.25% pancreatin digestion after kind in In 2 piece of 24 orifice plate, every hole kind 6 × 10⁴A cell, and DMEM culture medium of the 500 μ L containing 10%FBS is added.

Third step, transfection.2 piece of 24 orifice plate is divided into two groups.It arrives in for 24 hours in inoculating cell 16, is added in each group PWS46, pGY32, pGY102, pSZ69, pSZ83 and pSZ84 are turned with the ratio of 4:4:1:2:2:2 (w/w/w/w/w/w) and PEI The DMEM of transfection reagent and serum-free is mixed, and is dropped evenly after being stored at room temperature 15min into 24 well culture plates.The wherein preparation in every hole Total volume is 50 μ L, and plasmid and PEI mass ratio are 1:3.DMEM culture medium of the 500 μ L containing 10%FBS is changed to after transfection 6h to carry out Culture.

4th step, illumination.After changing liquid 14-18h, being placed in wavelength for first group is 730nm, intensity of illumination 1.5mW/cm² LED under illumination 20min, be immediately placed at dark and cultivate after illumination, continue illumination 20min after 48h, second group is first placed in Illumination 20min after the culture for 24 hours of dark place, be placed on culture at dark.

The results show that far-red light regulation the subgenomic transcription device based on CRISPR/Cas9 system have it is good can Inverse modulability.Experimental data is detailed in Figure of description 13, and all data are all presented in a manner of the independently duplicated experiment of n=3.

Case study on implementation 13

This example using TTN, RHOXF2, IL1RN, ASCL1 as reporter gene, verifying far-red light regulation based on CRISPR/ The subgenomic transcription device of Cas9 system activates the efficiency of multiple endogenous genes, but is not limited protection scope of the present invention System.Specific step is as follows:

Second step, inoculating cell.Will the good HEK-293 cell of growth conditions with 0.25% pancreatin digestion after kind in In 14 piece of 24 orifice plate, every hole kind 6 × 10⁴A cell, and DMEM culture medium of the 500 μ L containing 10%FBS is added.

Third step, transfection.14 piece of 24 orifice plate is divided into dark group and light group, every group is divided into 5 groups.It is thin in inoculation PWS46, pGY32, pGY102, pSZ69, pSZ83 and pSZ84 is added in for 24 hours in born of the same parents 16 in the first set, in the second set plus Enter pWS46, pGY32, pGY102, pSZ69, pSZ92 (P_U6- gRNA1 (IL1RN)-pA) and pSZ93 (P_U6-gRNA2(IL1RN)- PA), pWS46, pGY32, pGY102, pSZ69, pSZ105 (P are added in third group_U6- gRNA1 (RHOXF2)-pA) and pSZ106(P_U6- gRNA2 (RHOXF2)-pA), pWS46, pGY32, pGY102, pSZ69, pSZ103 is added in the 4th group (P_U6- gRNA1 (TTN)-pA) and pSZ104 (P_U6- gRNA2 (TTN)-pA), pWS46, pGY32 is added in the 5th group, pGY102,pSZ69、pSZ83、pSZ84、pSZ92(P_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), it mixes, is stored at room temperature with the DMEM of PEI transfection reagent and serum-free It is dropped evenly after 15min into 24 well culture plates.Wherein the preparation total volume in every hole is 50 μ L, and plasmid and PEI mass ratio are 1: 3.DMEM culture medium of the 500 μ L containing 10%FBS is changed to after transfection 6h to be cultivated.

4th step, illumination.After changing liquid 14-18h, it is 730nm, intensity of illumination 1.5mW/cm that light group, which is placed in wavelength,² LED under illumination 6h/d respectively, be immediately placed on dark at and cultivate after illumination, and dark organize then is placed in always training at dark It supports.

5th step, examining report gene.Real-time fluorescence quantitative PCR.

The results show that the subgenomic transcription device based on CRISPR/Cas9 system of far-red light regulation can simultaneously efficiently Activate multiple endogenous genes.Experimental data is detailed in Figure of description 14, and all data are all with the side of the independently duplicated experiment of n=3 Formula is presented.

1 plasmid construction table of table

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

CTCACGCTACGCTCA

The nucleotide sequence of SEQ ID NO.2:TATABox

TAGAGGGTATATAATGGAAGCTCG

SEQ ID NO.3: cytomegalovirus minimal promoter CMV_minNucleotide sequence

CCTGCAGGTCGAGCTCGGTACCCGGGTCGAGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTC GTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCC AGCCTCCGCG

SEQ ID NO.4: cytomegalovirus minimal promoter CMV_minMutant CMV_min3GNucleotide sequence AATGTCGAGGTAGGCGTGTACGGTGGGCGCCTATAAAAGCAGAGCTCGTTAGTGAACCGTCAGATCGCCTGGAGCAA TTCCACAACACTTTTGTCTTATACTTGGATCACC

SEQ ID NO.5:P_FRL1a:pA-3*whiG-P_hCMVmin

CAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTA TTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGC ATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAACCTCACGCTACGCTCACTCACGCTACGC TCACTCACGCTACGCTCACCTGCAGGTCGAGCTCGGTACCCGGGTCGAGTAGGCGTGTACGGTGGGAGGCCTATATA AGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCG GGACCGATCCAGCCTCCGCG

SEQ ID NO.6:P_FRL2a:3*whiG-P_hCMVmin

CTCACGCTACGCTCACTCACGCTACGCTCACTCACGCTACGCTCACCTGCAGGTCGAGCTCGGTACCCG GGTCGAGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGC CATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCG

SEQ ID NO.7:P_FRL3a:1*whiG-P_hCMVmin

CTCACGCTACGCTCACCTGCAGGTCGAGCTCGGTACCCGGGTCGAGTAGGCGTGTACGGTGGGAGGCCT ATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGA CACCGGGACCGATCCAGCCTCCGCG

SEQ ID NO.8:P_FRL1b:2*whiG-P_hCMVmin3G

CTCACGCTACGCTCACTCACGCTACGCTCACCTGCAGGATGTCGAGGTAGGCGTGTACGGTGGGCGCCT ATAAAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGCAATTCCACAACACTTTTGTCTTATACTTGGATC ACC

SEQ ID NO.9:P_FRL2b:3*whiG-P_hCMVmin3G

CTCACGCTACGCTCACTCACGCTACGCTCACTCACGCTACGCTCACCTGCAGGATGTCGAGGTAGGCGT GTACGGTGGGCGCCTATAAAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGCAATTCCACAACACTTTTG TCTTATACTTGGATCACC

The amino acid sequence of mutant (D10A, H840A) dCas9 of SEQ ID NO.10:Cas9 albumen MYPYDVPDYASPKKKRKVEASDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGE TAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPT IYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAI LSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYAD LFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDG GASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFT VYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHD LLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDK QSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQE LDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAE RGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIR KRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFD SPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKR MLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDK VLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLG GDSPKKKRKVEAS

SEQ ID NO.11: the transcription of Herpes simplex virus particles albumen VP16 transcriptional activation domain and its 4 copy Activation structure domain VP64

MGSGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLIN

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

GSPSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTPGPPQSL

SEQ ID NO.13: coat protein MS2

MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRKYTIKVEVPKVA TQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYSA

The fusion protein of SEQ ID NO.14:VP64-p65-Rta

GSGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLYIDSSGSPKKKRKV GPSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTPGPPQSLGSGSGSRDSREGMFLPKPEAGSAIS DVFEGREVCQPKRIRPFHPPGSPWANRPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEE TSQAVKALREMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEILDTFLNDECLL HAMHISTGLSIFDTSLF

SEQ ID NO.15: Features of The Heat Shock Transcription Factor HSF1 transcription activating domain

SAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSEFQQLLNQGVSMSHSTA EPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDEDFSSIADMDFSALLSQISSSGQGGGGSGFSVDTSA LLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPRPPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPV LFELGEGSYFSEGDGFAEDPTISLLTGSEPPKAKDPTVS

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

MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRKYTIKVEVPKVA TQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYSAGGGGSGGGGSGGGG SGPKKKRKVAAAMGSGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLIN

SEQ ID NO.17:FGTA2:MS2-Linker-NLS-VPR

MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRKYTIKVEVPKVA TQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYSAGGGGSGGGGSGGGG SGPKKKRKVAAAGSGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLYIDSSGSPKK KRKVGPSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTPGPPQSLGSGSGSRDSREGMFLPKPEAG SAISDVFEGREVCQPKRIRPFHPPGSPWANRPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLED PDEETSQAVKALREMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEILDTFLND ECLLHAMHISTGLSIFDTSLF

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

MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRKYTIKVEVPKVA TQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYSAGGGGSGGGGSGGGG SGPKKKRKVAAAGSPSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTPGPPQSLSAPVPKSTQAGE GTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSEFQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQR PPDPAPTPLGTSGLPNGLSGDEDFSSIADMDFSALLSQISSSGQGGGGSGFSVDTSALLDLFSPSVTVPDMSLPDLD SSLASIQELLSPQEPPRPPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAEDP TISLLTGSEPPKAKDPTVSMGSGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLIN

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

MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRKYTIKVEVPKVA TQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYSAGGGGSGGGGSGGGG SGPKKKRKVAAAGSPSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTPGPPQSLSAPVPKSTQAGE GTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSEFQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQR PPDPAPTPLGTSGLPNGLSGDEDFSSIADMDFSALLSQISSSGQGGGGSGFSVDTSALLDLFSPSVTVPDMSLPDLD SSLASIQELLSPQEPPRPPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAEDP TISLLTGSEPPKAKDPTVS

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

MASPKKKRKVEASSSEYAKQLGAKLRAIRTQQGLSLHGVEEKSQGRWKAVVVGSYERGDRAVTVQRLAE LADFYGVPVQELLPGTTPGGAAEPPPKLVLDLERLAHVPQEKAGPLQRYAATIQSQRGDYNGKVLSIRQDDLRTLAV IYDQSPSVLTEQLISWGVLDADARRAVAHEEN

The amino acid sequence of SEQ ID NO.21, nuclear localization signal (NLS)

TSPKKKRKVEDTS

The amino acid sequence of SEQ ID NO.22, linkage function peptide (Linker)

ASGSGGG

The sequence of photosensitive two guanylate cyclase (BphS) of SEQ ID NO.23 bacterium

MARGCLMTISGGTFDPSICEMEPIATPGAIQPHGALMTARADSGRVAHASVNLGEILGLPAASVLGAPI GEVIGRVNEILLREARRSGSETPETIGSFRRSDGQLLHLHAFQSGDYMCLDIEPVRDEDGRLPPGARQSVIETFSSA MTQVELCELAVHGLQLVLGYDRVMAYRFGADGHGEVIAERRRQDLEPYLGLHYPASDIPQIARALYLRQRVGAIADA CYRPVPLLGHPELDDGKPLDLTHSSLRSVSPVHLDYMQNMNTAASLTIGLADGDRLWGMLVCHNTTPRIAGPEWRAA AGMIGQVVSLLLSRLGEVENAAETLARQSTLSTLVERLSTGDTLAAAFVAADQLILDLVGASAAVVRLAGQELHFGR TPPVDAMQKVLDSLGRPSPLEVLSLDDVTLRHPELPELLAAGSGILLLPLTSGDGDLIAWFRPEHVQTITWGGNPAE HGTWNPATQRMRPRASFDAWKETVTGRSLPWTSAERNCARELGEAIAAEMAQRTRAEELERVAMVDSLTRLWNRLGI ETLLKREWEYATRKNSPISIVMIDFDNFKQINDQHGHLVGDEVLQGSARLIISVLASYDILGRWGGDEFMLILPGSG REQTAVLLERIQATIAQNPVPTSAGPMAISLSMGGVSVFTNQGEALQYWVEQADNQLMKVKRLGKGNFQLAEYHHHH HH

The amino acid sequence of the fusion protein p65-VP64-BldD of SEQ ID NO.24 downstream responses

ATMPSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTPGPPQSLMGSGRADALDDFDLD MLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLINASGSGGGGDVMASPKKKRKVEASSSEYAKQLGAK LRAIRTQQGLSLHGVEEKSQGRWKAVVVGSYERGDRAVTVQRLAELADFYGVPVQELLPGTTPGGAAEPPPKLVLDL ERLAHVPQEKAGPLQRYAATIQSQRGDYNGKVLSIRQDDLRTLAVIYDQSPSVLTEQLISWGVLDADARRAVAHEEN

Protection content of the invention is not limited to above embodiments.Without departing from the spirit and scope of the invention, originally Field technical staff it is conceivable that variation and advantage be all included in the present invention, and with appended claims be protect Protect range.

SEQUENCE LISTING

<110>East China Normal University

<120>a kind of far-red light regulates and controls subgenomic transcription device, construction method and application based on CRISPR/Cas9 system

<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 kind of subgenomic transcription device based on CRISPR/Cas9 system of far-red light regulation, which is characterized in that described device It include: far-red light primary element, far-red light response element and genome position transcriptional elements.

2. the subgenomic transcription device based on CRISPR/Cas9 system of far-red light regulation as described in claim 1, feature It is, the far-red light primary element includes the degrading enzyme YhjH of the photosensitive two guanylate cyclases BphS and c-di-GMP of bacterium.

3. the subgenomic transcription device based on CRISPR/Cas9 system of far-red light regulation as claimed in claim 2, feature It is, the amino acid sequence of the BphS is as shown in SEQ ID NO.23；The coding base of the degrading enzyme YhjH of the c-di-GMP Because of sequence Genebank accession number: ANK04038.

4. the subgenomic transcription device based on CRISPR/Cas9 system of far-red light regulation as described in claim 1, feature It is, the far-red light response element includes the fusion protein p65-VP64-BldD for responding c-di-GMP, and starting expression transcription swashs The promoter P of son living_FRLWith the transcription activator FGTAs of starting transcriptional activation downstream gene.

5. the subgenomic transcription device based on CRISPR/Cas9 system of far-red light regulation as claimed in claim 4, feature It is,

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

The promoter P_FRLIncluding the weak promoter that the protein bound DNA sequence dna of BldD and promotor gene are expressed, nucleotide sequence It is arbitrary a kind of selected from the sequence as shown in SEQ ID NO.5-9；The weak promoter of promotor gene expression includes TATAbox, cytomegalovirus hCMV minimal promoter and its mutant hCMVmin 3G promoter；

The transcription activator FGTAs includes the different combinations between MS2, VP64, p65, VPR, HSF1, amino acid sequence Arrange any one in sequence shown in the optional ID of SEQ freely NO.16-19.

6. the subgenomic transcription device based on CRISPR/Cas9 system of far-red light regulation as described in claim 1, feature Be, genome positioning transcriptional elements can efficient targeting to the site of genome target gene promoter, including dCas9 Albumen and with the single stranded RNA for instructing targeting；Wherein, the dCas9 is mutant D10A, the H840A of Cas9 albumen, Amino acid sequence is as shown in sequence SEQ ID NO.10.

7. a kind of construction method of the subgenomic transcription device based on CRISPR/Cas9 system of far-red light regulation, feature exist In, comprising the following steps:

(1) far-red light primary element is constructed

Building as the polypeptide of DNA binding domain and c-di-GMP binding domain, as the polypeptide of nuclear localization signal NLS, as connection Far-red light primary element of the complex of the polypeptide in domain and the polypeptide as transcriptional regulatory domain as system；

(2) far-red light response element is constructed；

(3) building genome positions transcriptional elements.

8. construction method as claimed in claim 7, which is characterized in that described to be used as DNA binding domain and c-di- in step (1) The polypeptide of GMP binding domain can be with the albumen in conjunction with specific DNA sequence dna, including BldD egg after in conjunction with c-di-GMP White, amino acid sequence is as shown in SEQ ID NO.20.

9. construction method as claimed in claim 7, which is characterized in that described as nuclear localization signal NLS's in step (1) Polypeptide can copy diversified forms for 1-3, and amino acid sequence is as shown in SEQ ID NO.21.

10. construction method as claimed in claim 7, which is characterized in that described as the more of linkage function domain in step (1) Peptide, length can be from 0-30 amino acid diversified forms, and amino acid sequence is as shown in SEQ ID NO.22.

11. a kind of kit, which is characterized in that the kit contain far-red light as described in claim 1 regulation based on The subgenomic transcription device of CRISPR/Cas9 system.

12. a kind of kit, which is characterized in that the kit contain far-red light as described in claim 1 regulation based on The carrier for expression of eukaryon of the subgenomic transcription device of CRISPR/Cas9 system and/or the place for having transfected the carrier for expression of eukaryon Chief cell and/or engineering cell transplantation carrier and corresponding specification.

13. the subgenomic transcription device based on CRISPR/Cas9 system of far-red light as described in claim 1 regulation or such as Kit described in claim 11 or 12 is in the application in induction endogenous genes of mammal expression.