CN110468122B

CN110468122B - Far-red light regulated stem cell differentiation induction system and application

Info

Publication number: CN110468122B
Application number: CN201810449225.3A
Authority: CN
Inventors: 叶海峰; 王美艳; 邵佳伟; 周阳
Original assignee: Suzhou Syntherapy Co ltd
Current assignee: Shanghai Zhennuo Biotechnology Co ltd
Priority date: 2018-05-11
Filing date: 2018-05-11
Publication date: 2023-04-07
Anticipated expiration: 2038-05-11
Also published as: CN110468122A

Abstract

The invention discloses a far-red light regulated and controlled system for inducing stem cell differentiation, which comprises a far-red light regulated and controlled gene expression loop control system and a gene locator. The invention also provides a far-red light regulated stem cell differentiation induction method, which can efficiently differentiate Induced Pluripotent Stem Cells (iPSCs) into mature nerve cells. The method can induce the stem cell differentiation by far-infrared light, has the characteristics of no toxicity, high efficiency, insulativity and flexibility, and has huge potential application value in the fields of cell therapy and regenerative medicine.

Description

Far-red light regulated stem cell differentiation induction system and application

Technical Field

The invention relates to the multidisciplinary crossing fields of synthetic biology, optogenetics, stem cells and the like, in particular to a method for inducing stem cell differentiation under far-red light regulation, efficient induction of stem cell differentiation and application of the method in stem cell differentiation research.

Background

Induced Pluripotent Stem Cells (iPSCs) are a great development of life science, solve the problems of immunological rejection and ethical ethics in the stem cell research field, can self-renew and maintain an undifferentiated state, can be directionally induced into various somatic cells in vitro, and have great application value in both theoretical research and clinical application. The induction of iPSCs to differentiate into different somatic cells has great significance in the research of disease models and mechanisms. At present, although there are many reports and studies on the induction of stem cell differentiation into adult cells, the studies on the regulation and control of stem cell differentiation into adult cells still need to be further improved, for example, the safety, non-toxicity, spatial specificity, flexible regulation and control, high efficiency of induction, etc. of the induction conditions are problems to be improved.

Far-red light is an ideal inducer of gene expression. Has the advantages of easy availability, space-time specificity, safety, no toxicity, and small irritation to cells. Therefore, far-red light is used as an inducer to regulate stem cell differentiation and has great application value in stem cell research.

Disclosure of Invention

The invention organically combines optogenetics, gene editing technology and regenerative medicine for the first time, provides a method for inducing stem cell differentiation by far-red light regulation and control, induces iPSCs to differentiate into somatic cells, can activate any exogenous and endogenous genes by far-red light, and can induce stem cells to differentiate into mature cells. The invention has the characteristics of good orthogonality, high stability, high induction efficiency, no toxic or side effect and the like, and has huge potential application value in the research of precisely and temporally regulating somatic cell behaviors and stem cells in the genetic engineering of mammals.

The invention provides a far-red light regulated system for inducing stem cell differentiation for the first time, which comprises a far-red light regulated gene expression loop control system (FACE system) and a gene locator.

The far-red light regulatory gene expression loop control system (FACE system) can efficiently differentiate induced pluripotent stem cells into mature nerve cells, provides a new tool for stem cell differentiation, can be used for research and treatment of related diseases, and has great potential application value.

Wherein the far-red light regulation and control system (FACE system) can efficiently induce the transcription expression of endogenous genes, and comprises bacterial photosensitive diguanylate cyclase BphS, degrading enzyme YhjH of c-di-GMP, activator P65-VP64-BldD expressed by BldD fusion, and promoter P combined with BldD recognition _FRL1b [(whiG) ₂ -P _hCMVmin3G ]And the sequence is shown in SEQ ID NO. 1-4.

The gene locator can effectively target to the promoter position of a target gene, and comprises dCas9 protein and all single-stranded RNA (sgRNA) with the function of guiding the target gene, wherein the dCas9 amino acid sequence is shown as SEQ ID NO. 5. The sequence of the single-stranded RNA is selected from all genome-matched sequences such as the sequence 6,7 (SEQ ID NO.6, 7).

The dCas9 protein can be combined with a transcription activator expressed by a far-red light regulatory gene expression loop control system, and the sgRNA is matched with a target gene locus to start the expression of any exogenous gene.

Furthermore, the dCas9 protein can also be combined with a transcription activator expressed by a far-red light regulatory gene expression loop control system, and the sgRNA is matched with a target gene promoter site to start the expression of any endogenous gene in vivo and in vitro; wherein the any endogenous gene in vivo comprises an endogenous gene associated with muscle in vivo.

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

The invention provides a method for inducing stem cell differentiation regulated by far-red light, which is artificially designed and synthesized.

The method comprises the following steps:

(1) Seeding cells

iPSCs with good growth state are inoculated into a 24-hole culture plate, and the culture medium in the culture plate is GMEM culture medium containing 15% fetal calf serum and 1% nonessential amino acid. (ii) a

Further, the culture medium further comprises a concentration of 10 ^-3 Glutamine of M, concentration 10 ^-3 Beta-mercaptoethanol of M, recombinant murine leukemia inhibitory factor with an activity of 1000U/mL, the medium being replaced daily.

(2) Production of stable transgenic plants

Electrically transferring the sleeking Beauty transposase expression vector into an iPSCs cell by using a far-red light regulatory gene expression loop control system (FACE system); and then, inoculating the iPSCs into a 24-pore plate, and screening out iPSCs stably expressing the far-red light regulatory gene expression loop control system by using puromycin and antibiotics.

Wherein the FACE system is pWS287 (ITR-P) _hCMV -p65-VP64-BldD-pA:P _hCMV -BphS-pA:P _mPGK -PuroR-pA-ITR)、pWS289(ITR-P _FRL1b -FGTA4-pA:P _mPGK -ZeoR-pA-ITR；P _FRL1b ,pA-(whiG) ₃ -P _hCMVmin (ii) a FGTA4, MS2-P65-HSF 1-ITR); the Sleeping Beauty transposase expression vector is pCMV-T7-SB100 (P) _hCMV -SB100X-pA)。

(3) Viral infection

Slow virus containing dCas9 and sgRNA, pWS74 (LTR-P) of related endogenous gene NEUROG2 for targeting differentiation into neurons _U6 -sgRNA1 (NEUROG 2) -LTR) and pWS76 (LTR-P) _U6 -sgRNA2(NEUROG2)-LTR)]Infecting iPSCs of the stable expression far-red light regulatory gene expression loop control system (FACE system), replacing a GMEM culture medium without LIF every day, and irradiating for 6h/d in far-red light.

Wherein the lentivirus containing dCas9 is pXS187 (LTR-P) _EF1a -dCas9-p2A-Blast-LTR)。

(4) Induced differentiation

The fifth day, 5X 10 cells were added to the medium ^-7 Trans RA of M, after day eight differentiated neural cells can be obtained.

The action mechanism of the far-red light regulated and controlled method for inducing stem cell differentiation is that when the bacterial photosensitive diguanylate cyclase BphS generates c-di-GMP under the far-red light irradiation condition, the c-di-GMP is combined with BldD to form a dimer, and transcription activator fused with dCas9 at the downstream of the transcription expression is induced, so that a specific transcription factor is activated, and the stem cell is induced to be directionally differentiated. When the illumination is stopped and the c-di-GMP cannot be generated, the synthesized c-di-GMP is degraded, the BldD cannot form a dimer, the dCas9 fused transcription activator cannot be expressed, the target transcription factor cannot be activated, and the stem cell cannot be differentiated.

The invention also provides a eukaryotic expression vector and an engineered cell of the method for inducing stem cell differentiation under far-red light regulation.

The invention also provides a method for preparing the eukaryotic expression vector, the engineered cell or the engineered cell transplantation vector containing the far-red light regulated and controlled method for inducing stem cell differentiation.

Wherein, the eukaryotic expression vector comprises a mammalian cell expression vector containing the far-red light regulated stem cell differentiation induction method. The expression vector can be a vector containing a far-red light regulation and control method coding gene alone or a vector containing a gene locator coding gene alone or both, and the construction modes of all the aforementioned mammalian cell expression vectors are detailed in table 1.

The invention also provides a construction method of the far-red light regulated stem cell differentiation induction system, which specifically comprises the following steps:

(1) And constructing a far-red light regulation gene expression loop control system.

Wherein, the far-red light regulation gene expression loop control system comprises far-red light sensing protein Bphs, the amino acid sequence is shown as SEQ ID NO.1, fusion protein P65-VP64-BldD responding to c-di-GMP, the amino acid sequence is shown as SEQ ID NO.3, and promoter P for starting expression of transcription activator _FRL The DNA sequence is shown in SEQ ID NO.4, and the transcription activator FGTA4 for starting transcription activation of downstream genes, and the amino acid sequence is shown in SEQ ID NO. 8.

(2) And constructing a gene locator.

Wherein, the gene localizer comprises dCas9 protein and sgRNA sequences of related endogenous gene NEUROG2 which is targeted to differentiate into neurons, and the sgRNA sequences are shown in SEQ ID NO.6 and 7.

The invention also provides application of the far-red light regulated system for inducing stem cell differentiation in regulating stem cell differentiation.

The beneficial effects of the invention include: can be used for the directional differentiation of stem cells, and has the characteristics of high induction efficiency, no toxic or side effect and the like. The method for inducing stem cell differentiation by far-red light regulation can quickly regulate endogenous genes by far-red light and induce stem cell differentiation, and has the characteristics of high induction efficiency, high space-time specificity, no toxic or side effect and the like. Has great potential application value in the research of precisely regulating the somatic cell behavior in time and space in the genetic engineering of mammals and stem cells.

Drawings

FIG. 1 is a comparison graph of far-red light-regulated effects of inducible activation of the Lama1 gene and blue light;

FIG. 2 is a graph comparing the effects of far-red light-regulated induced activation of Fst gene with blue light;

FIG. 3 is a schematic diagram of a far-red light-regulated method for inducing stem cell differentiation;

FIG. 4 is a diagram showing the results of gene expression of activating genes in a method for inducing stem cell differentiation under far-red light regulation;

FIG. 5 is a diagram showing the results of marker genes differentiated into neurons by a method for inducing stem cell differentiation regulated by far-red light;

fig. 6 is a graph of calcium ion imaging results.

Detailed Description

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

Materials and methods

Molecular cloning

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

All primers used for PCR were synthesized by Kingzhi Biotech, inc. The expression plasmids constructed in the examples of the present invention were subjected to sequence determination, which was accomplished by Jinzhi Biotechnology, inc. Phanta Max Super-Fidelity DNA polymerase used in the examples of the present invention was purchased from Biotech, inc. of Nanjing Novowed Toxa. The endonuclease and the T4 DNA ligase were purchased from TaKaRa. Homologous recombinases were purchased from Heyu Biotechnology (Shanghai) Ltd. Phanta Max Super-Fidelity DNA polymerase was purchased with the corresponding polymerase buffer and dNTPs. The endonuclease, T4 DNA ligase and homologous recombinase were purchased with the corresponding buffer. Yeast Extract (Yeast Extract), tryptone (Trypton), agar powder, and ampicillin (Amp) were obtained from Shanghai Biotech, inc. DNAMarker DL5000, DNA Marker DL2000 (baobao bioengineering ltd); nucleic acid dye EB (guangdong national ao biotechnology); a plasmid small extraction kit (Tiangen Biochemical technology (Beijing) Co., ltd.); the DNA gel recovery kit and the PCR product purification kit are purchased from Shikoku Biotechnology GmbH; the other reagents such as absolute ethyl alcohol, naCl and the like mentioned in the examples are all domestic analytical pure products. The method comprises the following steps of (1) glue recovery and purification recovery of DNA fragments, wherein the steps are carried out according to the operation instructions of a DNA glue recovery kit and a PCR product purification kit (Kangji Biotech Co., ltd.); plasmid extraction procedure was performed according to the plasmid Mini-extraction kit (Tiangen Biochemical technology (Beijing) Ltd.).

Cell culture and transfection

The following cell line and PEI transfection are used as examples to illustrate the working of the far-red light-regulated method for inducing stem cell differentiation in cells, but do not limit the scope of the present invention.

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

Culture of mouse iPSCs

Mouse Induced Pluripotent Stem Cells (iPSCs) were cultured in GMEM medium (GMEM, gibco; cat. No. 11710-035) medium supplemented with 15% (v/v) fetal bovine serum (FBS, gibco; cat. No. 16000-044), 1% (v/v) nonessential amino acids (Gibco; cat. No. 11140-050), 1X 10 ^-3 M Glutamine (Gibco; cat. No. 35050-061), 0.1X 10 ^-3 M beta-mercaptoethanol (Sigma; cat. No. M3148) and 1000U/mL recombinant mouse leukemia inhibitory factor (LIF, millipore; cat. No. ESG1107). The medium was changed daily.

Real-time fluorescent quantitative PCR analysis

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

Lentiviral production

The seeds were grown at 5X 10 in 15cm diameter petri dishes ⁶ A2000. Mu.l mixed system of 3. The virus was collected 48h after transfection, filtered through a 0.45m PVDF filter and stored at-80 ℃.

Calcium ion imaging

Differentiated neural cells were washed three times with Hanks balanced salt solution (1 × HBSS) and then treated with 3 μ M calcium indicator Fluo-4AM buffer solution (cat. No. f 14201) at 37 ℃ for 60min. Excess dye was removed and washed three times with 1 × HBSS. These cells were placed in 50mM KCl solution and imaged for 5 minutes with a Leica DMI8 microscope (Wetzlar, germany) with a 5 minute delay at an excitation wavelength of 488nm and the data analyzed using Leica LAS AF software.

Example 1

In the embodiment, the Lama1 gene is taken as an experimental object to verify the contrast effect of the far-red light regulation and control gene expression loop control system on activating endogenous genes and blue light, but the protection scope of the invention is not limited. The method comprises the following specific steps:

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

And secondly, electrically switching. The CPTS 2.0 system (pWS 303 (P) _hCMV -MS2-NES-CIBN-pA)、pWS304(P _hCMV -CRY2PHR-NLS-p65-HSF1-pA)、pGY174[P _U6 -sgRNA1(Lama1)-pA-P _U6 -sgRNA2(Lama1)-pA-P _hCMV -dCas9-pA]) And withThe far-red light regulatory gene expression loop control system (pWS 189/pGY32/pGY102/pGY 174) was electrotransferred into the mouse posterior tibial muscle.

And thirdly, illuminating. The electroporated mice were exposed to blue and far red light (10 mW/cm 2) for 4h daily.

And fourthly, detecting. Three days later, the mouse tibialis posterior muscle was collected and subjected to qPCR detection.

The result shows that blue light can not activate endogenous genes, and the far-red light regulation gene expression loop control system can effectively activate endogenous genes. Experimental data the results are shown in figure 1 of the specification, and all data are presented as n =6 independent replication experiments

Example 2

In the example, the Fst gene is taken as an experimental object to verify the comparison effect of the far-red light regulatory gene expression loop control system on activating the endogenous gene and blue light, but the protection scope of the invention is not limited. The method comprises the following specific steps:

And secondly, electrically switching. The CPTS 2.0 system (pWS 303, pWS304, pGY 177) and the far-red light regulatory gene expression loop control system (pWS 189, pGY32, pGY102, pGY 177) were electrotransferred into the mouse posterior tibialis.

And thirdly, illuminating. The electroporated mice were exposed to blue and far-red light (10 mW/cm) ² ) The lower part is illuminated for 4 hours every day.

And fourthly, detecting. Three days later, the mouse posterior tibial muscle was collected and subjected to qPCR detection.

The result shows that the blue light can not activate the endogenous gene, and the far-red light regulation gene expression loop control system can effectively activate the endogenous gene. Experimental data the results are shown in fig. 2 of the specification, and all data are presented as n =5 independent replication experiments

Embodiment 3

The example uses mouse induced pluripotent stem cells stably expressing a FACE system as an experimental object to verify that the far-red light-regulated method for inducing stem cell differentiation differentiates into nerve cells, but does not limit the protection scope of the invention. The method comprises the following specific steps:

In the second step, cells are seeded. Mouse induced pluripotent stem cells stably expressing the FACE system were seeded on two 24-well gel-coated cell culture plates, 1. Mu.g/ml puromycin and 100. Mu.g/ml bleomycin were added to the medium, and the cells were cultured at 37 ℃ in an incubator containing 5% carbon dioxide

And thirdly, infection. The 2 24-well plates were divided into a dark group and a light group. 24h after seeding the cells with a medium containing dCas9[ pXS187 (LTR-P) _EF1a -dCas9-p2A-Blast-LTR)]and 2sgRNAs[pWS74(LTR-P _U6 -sgRNA1(NEUROG2)-LTR),pWS76(LTR-P _U6 -sgRNA2(NEUROG2)-LTR)]Lentiviruses targeting the NEUROG2 gene infect cells. The first four days, the cells were changed with medium every day, and the following four days, 5X 10 cells were added to the medium ^-7 RA of M (Sigma; cat. No. R2625)

And fourthly, illuminating. The dark group was kept in the dark for culture, and the light group was irradiated with far-red light for 6 hours per day.

Fifthly, detecting the expression quantity of the target gene by using real-time fluorescent quantitative PCR (polymerase chain reaction) on

days

1, 4 and 8 respectively; and detecting the nerve cell marker gene by real-time fluorescence quantitative PCR on the eighth day, and performing a calcium ion imaging experiment.

The result shows that in the method based on the CRISPR/Cas9 system for far-red light regulation, when the promoter is P _FRL1b [(whiG) ₂ -P _hCMVmin3G ；pGY102]The activation efficiency is most pronounced. Experimental data the results are illustrated in the accompanying figures 3, 4, 5, 6 of the specification, all data being presented as n =3 independent replicate experiments.

TABLE 1 plasmid construction

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SEQ ID NO.1 sequence of bacterial photosensitive diguanylate cyclase (BphS)

MARGCLMTISGGTFDPSICEMEPIATPGAIQPHGALMTARADSGRVAHASVNLGEILGLPAASVLGAPIGEVIGRVNEILLREARRSGSETPETIGSFRRSDGQLLHLHAFQSGDYMCLDIEPVRDEDGRLPPGARQSVIETFSSAMTQVELCELAVHGLQLVLGYDRVMAYRFGADGHGEVIAERRRQDLEPYLGLHYPASDIPQIARALYLRQRVGAIADACYRPVPLLGHPELDDGKPLDLTHSSLRSVSPVHLDYMQNMNTAASLTIGLADGDRLWGMLVCHNTTPRIAGPEWRAAAGMIGQVVSLLLSRLGEVENAAETLARQSTLSTLVERLSTGDTLAAAFVAADQLILDLVGASAAVVRLAGQELHFGRTPPVDAMQKVLDSLGRPSPLEVLSLDDVTLRHPELPELLAAGSGILLLPLTSGDGDLIAWFRPEHVQTITWGGNPAEHGTWNPATQRMRPRASFDAWKETVTGRSLPWTSAERNCARELGEAIAAEMAQRTRAEELERVAMVDSLTRLWNRLGIETLLKREWEYATRKNSPISIVMIDFDNFKQINDQHGHLVGDEVLQGSARLIISVLASYDILGRWGGDEFMLILPGSGREQTAVLLERIQATIAQNPVPTSAGPMAISLSMGGVSVFTNQGEALQYWVEQADNQLMKVKRLGKGNFQLAEYHHHHHH

SEQ ID NO.2c-di-GMP degrading enzyme (YhjH) amino acid sequence

MIRQVIQRISNPEASIESLQERRFWLQCERAYTWQPIYQTCGRLMAVELLTVVTHPLNPSQRLPPDRYFTEITVSHRMEVVKEQIDLLAQKADFFIEHGLLASVNIDGPTLIALRQQPKILRQIERLPWLRFELVEHIRLPKDSTFASMCEFGPLWLDDFGTGMANFSALSEVRYDYIKIARELFVMLRQSPEGRTLFSQLLHLMNRYCRGVIVEGVETPEEWRDVQNSPAFAAQGWFLSRPAPIETLNTAVLAL

Amino acid sequence of activator p65-VP64-BldD fused with SEQ ID NO.3BldD

ATMPSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTPGPPQSLMGSGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLINASGSGGGGDVMASPKKKRKVEASSSEYAKQLGAKLRAIRTQQGLSLHGVEEKSQGRWKAVVVGSYERGDRAVTVQRLAELADFYGVPVQELLPGTTPGGAAEPPPKLVLDLERLAHVPQEKAGPLQRYAATIQSQRGDYNGKVLSIRQDDLRTLAVIYDQSPSVLTEQLISWGVLDADARRAVAHEEN

SEQ ID NO.4：PFRL1b:2*whiG-PhCMVmin3G

CTCACGCTACGCTCACTCACGCTACGCTCACCTGCAGGATGTCGAGGTAGGCGTGTACGGTGGGCGCCTATAAAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGCAATTCCACAACACTTTTGTCTTATACTTGGATCACC

SEQ ID No.5: amino acid sequence of mutant (D10A, H840A) dCas9 of Cas9 protein

MYPYDVPDYASPKKKRKVEASDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSPKKKRKVEAS

SEQ ID NO.6 sgRNA1(NEUROG2)

GTGGATGGCCAGGCCAGGGGA

SEQ ID NO.7sgRNA2(NEUROG2)

GCTGCAGCCTGGAGCGCCAAC

SEQ ID NO.8：FGTA4:MS2-Linker-NLS-p65-HSF1

MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYSAGGGGSGGGGSGGGGSGPKKKRKVAAAGSPSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTPGPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSEFQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDEDFSSIADMDFSALLSQISSSGQGGGGSGFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPRPPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAEDPTISLLTGSEPPKAKDPTVS

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

SEQUENCE LISTING

<110> Suzhou Xinsai Biotechnology Ltd

<120> far-red light regulated stem cell differentiation induction system and application

<160> 38

<170> PatentIn version 3.3

<210> 1

<211> 687

<212> PRT

<213> Artificial sequence

<400> 1

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> 2

<211> 255

<212> PRT

<213> Artificial sequence

<400> 2

Met Ile Arg Gln Val Ile Gln Arg Ile Ser Asn Pro Glu Ala Ser Ile

1 5 10 15

Glu Ser Leu Gln Glu Arg Arg Phe Trp Leu Gln Cys Glu Arg Ala Tyr

20 25 30

Thr Trp Gln Pro Ile Tyr Gln Thr Cys Gly Arg Leu Met Ala Val Glu

35 40 45

Leu Leu Thr Val Val Thr His Pro Leu Asn Pro Ser Gln Arg Leu Pro

50 55 60

Pro Asp Arg Tyr Phe Thr Glu Ile Thr Val Ser His Arg Met Glu Val

65 70 75 80

Val Lys Glu Gln Ile Asp Leu Leu Ala Gln Lys Ala Asp Phe Phe Ile

85 90 95

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

100 105 110

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

115 120 125

Trp Leu Arg Phe Glu Leu Val Glu His Ile Arg Leu Pro Lys Asp Ser

130 135 140

Thr Phe Ala Ser Met Cys Glu Phe Gly Pro Leu Trp Leu Asp Asp Phe

145 150 155 160

Gly Thr Gly Met Ala Asn Phe Ser Ala Leu Ser Glu Val Arg Tyr Asp

165 170 175

Tyr Ile Lys Ile Ala Arg Glu Leu Phe Val Met Leu Arg Gln Ser Pro

180 185 190

Glu Gly Arg Thr Leu Phe Ser Gln Leu Leu His Leu Met Asn Arg Tyr

195 200 205

Cys Arg Gly Val Ile Val Glu Gly Val Glu Thr Pro Glu Glu Trp Arg

210 215 220

Asp Val Gln Asn Ser Pro Ala Phe Ala Ala Gln Gly Trp Phe Leu Ser

225 230 235 240

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

245 250 255

<210> 3

<211> 300

<212> PRT

<213> Artificial sequence

<400> 3

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> 4

<211> 149

<212> DNA

<213> Artificial sequence

<400> 4

ctcacgctac gctcactcac gctacgctca cctgcaggat gtcgaggtag gcgtgtacgg 60

tgggcgccta taaaagcaga gctcgtttag tgaaccgtca gatcgcctgg agcaattcca 120

caacactttt gtcttatact tggatcacc 149

<210> 5

<211> 1399

<212> PRT

<213> Artificial sequence

<400> 5

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> 6

<211> 21

<212> DNA

<213> Artificial sequence

<400> 6

gtggatggcc aggccagggg a 21

<210> 7

<211> 21

<212> DNA

<213> Artificial sequence

<400> 7

gctgcagcct ggagcgccaa c 21

<210> 8

<211> 473

<212> PRT

<213> Artificial sequence

<400> 8

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> 9

<211> 48

<212> DNA

<213> Artificial sequence

<400> 9

tttaaactta agcttggtac gccaccatgt acccatacga tgttccag 48

<210> 10

<211> 48

<212> DNA

<213> Artificial sequence

<400> 10

ggtttaaacg ggccctctag ttagctggcc tccacctttc tcttcttc 48

<210> 11

<211> 51

<212> DNA

<213> Artificial sequence

<400> 11

gatagtgctg gtagtgctgg tagtgctggt ggctccgggc gcgccgacgc g 51

<210> 12

<211> 47

<212> DNA

<213> Artificial sequence

<400> 12

gatccgagct cggtaccaag cttttagttt tcctcgtgag ccacagc 47

<210> 13

<211> 48

<212> DNA

<213> Artificial sequence

<400> 13

cttaagcttg gtaccgccac catgccttca gggcagatca gcaaccag 48

<210> 14

<211> 52

<212> DNA

<213> Artificial sequence

<400> 14

accagcacta ccagcactac cagcactatc cagtgactgg ggtggtcctg gg 52

<210> 15

<211> 50

<212> DNA

<213> Artificial sequence

<400> 15

gtcttatact tggatcaccg aattcgccac catggcttca aactttactc 50

<210> 16

<211> 54

<212> DNA

<213> Artificial sequence

<400> 16

ccagagctgt tttaaaagct ttaggagaca gtggggtcct tggctttggg aggc 54

<210> 17

<211> 52

<212> DNA

<213> Artificial sequence

<400> 17

ggaggcctag gcttttgcaa aaagcttgcc accatggcta gaggctgcct ca 52

<210> 18

<211> 53

<212> DNA

<213> Artificial sequence

<400> 18

gaagcggccg gccgccccga ctctagagtg gtgatggtgg tggtggtact cgg 53

<210> 19

<211> 29

<212> DNA

<213> Artificial sequence

<400> 19

ctagctagcc gagggcctat ttcccatga 29

<210> 20

<211> 28

<212> DNA

<213> Artificial sequence

<400> 20

ccggaattca taccgcacag atgcgtaa 28

<210> 21

<211> 34

<212> DNA

<213> Artificial sequence

<400> 21

cgcggatccg agggcagagg aagtctgcta acat 34

<210> 22

<211> 35

<212> DNA

<213> Artificial sequence

<400> 22

ccggaattct tagccctccc acacataacc agagg 35

<210> 23

<211> 38

<212> DNA

<213> Artificial sequence

<400> 23

ccgctcgagc ggtaaggggt ccgctatcta gagttgac 38

<210> 24

<211> 34

<212> DNA

<213> Artificial sequence

<400> 24

ctagctagct tagtggtgat ggtggtggtg gtac 34

<210> 25

<211> 25

<212> DNA

<213> Artificial sequence

<400> 25

cgacgcgtac ctgacgtccg atcca 25

<210> 26

<211> 30

<212> DNA

<213> Artificial sequence

<400> 26

ccgctcgaga gagctgtttt aaaagcttta 30

<210> 27

<211> 32

<212> DNA

<213> Artificial sequence

<400> 27

ccggaattcg agggcctatt tcccatgatt cc 32

<210> 28

<211> 31

<212> DNA

<213> Artificial sequence

<400> 28

cgcggatcca ttcaaaaaaa gcaccgactc g 31

<210> 29

<211> 29

<212> DNA

<213> Artificial sequence

<400> 29

cgggatccct tttgctggcc ttttgctca 29

<210> 30

<211> 31

<212> DNA

<213> Artificial sequence

<400> 30

cgacgcgtgc gggtgtcggg gctggcttaa c 31

<210> 31

<211> 47

<212> DNA

<213> Artificial sequence

<400> 31

gcgtttaaac ttaagcttgg taccgccacc atggcttcaa actttac 47

<210> 32

<211> 46

<212> DNA

<213> Artificial sequence

<400> 32

cttctcagtc tctcggggta caggctacct cctcctccgc ttcctc 46

<210> 33

<211> 46

<212> DNA

<213> Artificial sequence

<400> 33

gaggaagcgg aggaggaggt agcctgtacc ccgagagact gagaag 46

<210> 34

<211> 43

<212> DNA

<213> Artificial sequence

<400> 34

ggtttaaacg ggccctctag attagatgta gtcggtcttc tcg 43

<210> 35

<211> 44

<212> DNA

<213> Artificial sequence

<400> 35

gtttaaactt aagcttggta cctctggcgc caccatggcc agtc 44

<210> 36

<211> 46

<212> DNA

<213> Artificial sequence

<400> 36

ctcctccgct tccacctcct ccgggagcgg cgccgatcat gatctg 46

<210> 37

<211> 46

<212> DNA

<213> Artificial sequence

<400> 37

cagatcatga tcggcgccgc tcccggagga ggtggaagcg gaggag 46

<210> 38

<211> 44

<212> DNA

<213> Artificial sequence

<400> 38

ggtttaaacg ggccctctag attaggagac agtggggtcc ttgg 44

Claims

1. A far-red light regulated system for inducing stem cell differentiation, the system comprising: a far-red light regulation gene expression loop control system and a gene locator; the far-red light regulation gene expression loop control system can induce the transcription expression of genes;

the genes comprise bacterial photosensitive diguanylate cyclase BphS, degrading enzyme YhjH of c-di-GMP, activator expressed by BldD fusion, and promoter P combined with BldD recognition _FRL1b [(whiG) ₂ -P _hCMVmin3G ]；

The amino acid sequence of the bacterial photosensitive diguanylate cyclase BphS is shown as SEQ ID NO. 1; the amino acid sequence of the degrading enzyme YhjH of the c-di-GMP is shown as SEQ ID NO. 2; the amino acid sequence of the activator expressed by BldD fusion is shown in SEQ ID NO. 3; the BldD recognizes the bound promoter P _FRL1b [(whiG) ₂ -P _hCMVmin3G ]The DNA sequence of (A) is shown in SEQ ID NO. 4;

the localizer can effectively target to the promoter position of a target gene, including dCas9 protein and all single-stranded RNA with the function of guiding targeting, activate the expression of the target gene and induce the differentiation of stem cells;

the dCas9 protein can be combined with a transcription activator expressed by a far-red light regulatory gene expression loop control system, and the sgRNA is matched with a target gene locus to start the expression of any exogenous gene; wherein the amino acid sequence of the dCas9 protein is shown in SEQ ID NO. 5;

the dCas9 protein can be combined with a transcription activator expressed by a far-red light regulatory gene expression loop control system, and the sgRNA is matched with a target gene promoter site to start the expression of any endogenous gene in vivo and in vitro.

2. The system of claim 1, wherein the any endogenous gene in vivo comprises a muscle-related endogenous gene in vivo.

3. A method for using the far-red light-regulated stem cell differentiation induction system according to claim 1, comprising the following steps:

(1) Seeding cells

Will grow in good conditioniPSCs cells were seeded in 24-well culture plates; the culture medium in the culture plate is GMEM culture medium containing 15% of fetal calf serum and 1% of non-essential amino acid; the culture medium also comprises a concentration of 10 ^-3 Glutamine of M, concentration 10 ^-3 Beta-mercaptoethanol of M, recombinant mouse leukemia inhibitory factor with 1000U/mL activity, the culture medium needs to be replaced every day;

(2) Production of stable transgenic plants

Electrically transferring the iPSCs by a far-red light regulatory gene expression loop control system and a Sleeping Beauty transposase expression vector; then, inoculating the iPSCs into a 24-hole culture plate, and screening out iPSCs stably expressing a far-red light regulatory gene expression loop control system by using puromycin and antibiotics;

(3) Viral infection

A lentiviral vector containing dCas9 and sgRNA targeting the endogenous gene NEUROG2 for differentiation into neurons, pWS74: LTR-P _U6 -sgRNA1 (NEUROG 2) -LTR and pWS76: LTR-P _U6 -sgRNA2 (NEUROG 2) -LTR infects iPSCs cells stably expressing the far-red light regulatory gene expression loop control system, replacing the LIF-free GMEM medium every day, and irradiating for 6h/d in far-red light;

(4) Inducing differentiation.

4. A method for constructing the far-red light-regulated system for inducing stem cell differentiation according to claim 1, which comprises the following steps:

(1) Constructing a far-red light regulation gene expression loop control system;

(2) And constructing a gene locator.

5. Use of the far-red light-regulated stem cell differentiation-inducing system according to claim 1 for regulating stem cell differentiation.

6. A method for preparing a eukaryotic expression vector, an engineered cell or an engineered cell transplantation vector containing the far-red light-regulated stem cell differentiation-inducing system according to claim 1.