A light-controlled CRISPRi gene regulation system, its construction method and application
The CRISPRi system, which uses red and green light to regulate the light, solves the problems of large cell damage and small differences in target gene expression in existing technologies. It achieves efficient and precise regulation of target gene expression and is applicable to the fields of organism behavior control, industrial fermentation and medical health.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-09-29
- Publication Date
- 2026-03-10
AI Technical Summary
Existing light-controlled CRISPRi technology suffers from significant cell damage and small fold differences in target gene expression when regulating gene expression. In particular, it is harmful to the health of cells and operators under blue light irradiation, and the design concept generally results in a fold difference of no more than 10 times.
A light-controlled CRISPRi system, which utilizes red and green light to regulate the expression of CRISPRi, was designed by combining a light control module and a CRISPRi module. The system uses red and green light to silence or express CRISPRi, respectively. It includes a biological circuit and a light source device. The light control module adjusts the expression of the CRISPRi module according to the intensity and time of red and green light, thereby achieving precise regulation of the target gene.
It achieved approximately 40-fold difference in the expression of the target gene, reduced cell damage, improved the sensitivity and accuracy of the system, and reduced the risk of photodamage to cells.
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Figure CN115960864B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of gene regulation, in particular to a light-controlled CRISPRi gene regulation system, a construction method thereof and an application thereof. BACKGROUND
[0002] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology can use a complex of Cas9 protein and sgRNA to find and remove a target DNA sequence. CRISPRi (CRISPR interference) technology modifies the Cas9 protein into a dCas9 protein, so that the complex can silence various genes in animal, plant or microbial cells, thereby changing the life activities of the organism. After combining optogenetics technology with CRISPRi, different colors of light can be used to turn on or off CRISPRi, and then the life activities are regulated by light. This technology has a broad application prospect in the fields of biological behavior control, industrial fermentation and medical health.
[0003] Existing light-controlled CRISPRi technology mostly adopts the design idea of splitting dCas9 protein and the blue light-activated photosensitive element, and most of them are applied to eukaryotic cells.
[0004] Using a blue light-sensitive biological element to regulate CRISPRi means that the organism needs to be exposed to blue light for a long time. Blue light has a shorter wavelength (400-500 nm) and higher energy, which has been proven to cause cell damage. In addition, in the application scenarios such as fermentation production or medical experiments, blue light will damage the vision and health of the operator.
[0005] In order to realize the off / on of the light-controlled CRISPRi system, the design needs to meet the following conditions: under the first light condition (A), CRISPRi has no activity; under the second light condition (B), CRISPRi has activity, and the higher the ratio of the reporter gene (A / B) in the two states, the more sensitive the system, that is, the larger the difference multiple. Most of the current domestic and foreign researches adopt the design idea of "splitting-polymerization", which splits the dCas9 protein into two halves and connects light-controlled polymerization tags to each half. Due to the reasons such as the activity of the split state or the low polymerization efficiency, the light-controlled CRISPRi system using this design generally has a difference multiple of no more than 10 times.
[0006] Therefore, there is an urgent need for a light-controlled CRISPRi gene regulation system with small cell damage and large target gene expression difference multiple, a construction method thereof and an application thereof. SUMMARY
[0007] The present application aims to overcome the deficiencies of the prior art and provide a light-controlled CRISPRi gene regulation system, a construction method thereof and an application thereof.
[0008] To achieve the above-mentioned purpose, the technical solution adopted by the present application is as follows:
[0009] The present application provides a light-controlled CRISPRi gene regulation system, which comprises a biological circuit and a light source device.
[0010] The light control module adjusts the expression of the CRISPRi module according to the intensity and time of red and green light irradiation.
[0011] The CRISPRi module can inhibit any target gene in the cell after expression.
[0012] The light-controlled CRISPRi gene regulation system of the present application comprises a biological circuit responding to red and green light and a corresponding light source device.
[0013] As a preferred embodiment of the light-controlled CRISPRi gene regulation system of the present application, the light control module is a optogenetic gene expression control module.
[0014] The light control module designed by the present application can adjust the expression of the CRISPRi module according to the intensity and time of red and green light irradiation.
[0015] As a preferred embodiment of the light-controlled CRISPRi gene regulation system of the present application, the light control module comprises a PcpcG2-172 promoter and an RBS (ribosome binding site).
[0016] This invention optimizes the promoter and RBS of the light-controlled module, making the light-controlled CRISPRi gene regulation system more sensitive and enabling it to perform its corresponding functions.
[0017] Under red light irradiation, CcaS and CcaR remain unphosphorylated, the PcpcG2-172 promoter does not transcribe dCas9 and sgRNA, CRISPRi remains silent, and the target gene can be expressed normally. Under green light irradiation, CcaS transfers a phosphate group to CcaR, the PcpcG2-172 promoter transcribes dCas9 and sgRNA, CRISPRi begins to express, and at this point, the target gene is suppressed by CRISPRi and cannot be expressed.
[0018] In a preferred embodiment of the light-controlled CRISPRi gene regulation system of the present invention, the CRISPRi module is a dCas9-sgRNA gene inhibition module.
[0019] As a preferred embodiment of the light-controlled CRISPRi gene regulation system of the present invention, the CRISPRi module can change the target gene of the light-controlled CRISPRi gene regulation system simply by changing the target sequence of the sgRNA.
[0020] This invention allows for the alteration of the target gene in the light-controlled CRISPRi gene regulation system through a single PCR reaction by changing the target sequence of the sgRNA. After co-transforming the light-controlled module and the CRISPRi module into E. coli cells, applying light to the bacterial culture or agar plate can activate or deactivate the target gene, making its expression more controllable and possessing spatiotemporal specificity.
[0021] In a preferred embodiment of the light-controlled CRISPRi gene regulation system of the present invention, the emission wavelengths of red and green light are wavelengths that can induce optogenetic molecular responses.
[0022] In some embodiments, the wavelength of the red light is 650-680 nm, preferably 672 nm.
[0023] In some embodiments, the wavelength of the green light is 515-545 nm, preferably 535 nm.
[0024] In a preferred embodiment of the light-controlled CRISPRi gene regulation system of the present invention, the light control module is a plasmid or DNA sequence as shown in SEQ NO:1.
[0025] In a preferred embodiment of the light-controlled CRISPRi gene regulation system of the present invention, the CRISPRi module is a plasmid or DNA sequence as shown in SEQ NO:2.
[0026] The second object is to provide a construction method of the light-controlled CRISPRi gene regulation system, comprising the following steps: integrating related genes on a biological circuit, the biological circuit comprising a light-controlled module and a CRISPRi module, integrating the light-controlled module and the CRISPRi module into cells, and simultaneously constructing a light source device to form the light-controlled CRISPRi gene regulation system.
[0027] The light-controlled CRISPRi gene regulation system integrates all related genes on a biological circuit, the construction method of the light-controlled CRISPRi gene regulation system optimizes the promoter and RBS of the light-controlled module, so that the light-controlled CRISPRi gene regulation system has higher sensitivity, and the optimized light-controlled module is fused with the CRISPRi module, and a special light source device is designed and built. The light-controlled module can regulate dCas9 and sgRNA at the transcription level, so that CRISPRi is silenced under red light and expressed under green light, without involving the splitting and polymerization of dCas9 protein, so that the light-controlled CRISPRi gene regulation system constructed by the application can cause about 40 times difference in the expression of target genes, which is much higher than other light-controlled CRISPRi systems.
[0028] The third object is to provide an application of the light-controlled CRISPRi gene regulation system in opening or closing the expression of target genes in host cells.
[0029] Compared with the prior art, the application has the following beneficial effects:
[0030] The application provides a light-controlled CRISPRi gene regulation system, comprising a biological circuit and a light source device, when the light-controlled module is irradiated by red light, the CRISPRi module is silenced, and the gene targeted by CRISPRi is normally expressed; when the light-controlled module is irradiated by green light, the CRISPRi module is expressed, and the gene targeted by CRISPRi is silenced. The application introduces red and green light to accurately regulate CRISPRi, so that the opening and closing have spatiotemporal specificity. The application helps to deepen the understanding of light-controlled gene circuits, and has important guiding significance for constructing large-scale synthetic biology systems that can target multiple genes. In practical applications, the light-controlled CRISPRi gene regulation system can be integrated into cells, so that the expression of specific genes can be opened or closed in real time according to the needs, the cell behavior or metabolic pathway is regulated, the production cost is saved, and the production efficiency is improved. BRIEF DESCRIPTION OF DRAWINGS
[0031] Figure 1 Principle of the light-controlled CRISPRi gene regulation system Figure 1 A is a plasmid map; Figure 1 B is a working principle diagram of related genes in cells.
[0032] Figure 2 Figure for the results of the light-controlled CRISPRi gene regulation system to inhibit the genes downstream of the arabinose promoter in Example 2;
[0033] Figure 3 Figure for the results of the light-controlled CRISPRi gene regulation system to inhibit the β-galactosidase gene LacZ in Example 3;
[0034] Figure 4 Figure for the results of the light-controlled CRISPRi gene regulation system to inhibit FtsZ in Example 4;
[0035] Figure 5 Figure for the results of the light-controlled CRISPRi gene regulation system to inhibit MreB in Example 5. DETAILED DESCRIPTION
[0036] For the purpose of better illustrating the object, technical scheme and advantages of the present application, the present application will be further described below in conjunction with the drawings and specific examples.
[0037] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available unless otherwise specified.
[0038] Example 1, a light-controlled CRISPRi gene regulation system
[0039] A light-controlled CRISPRi gene regulation system comprises a biological circuit and a light source device; the biological circuit comprises a light control module and a CRISPRi module; the light source device is an electronic device emitting a certain range of wavelength of red light (672 nm) or green light (535 nm);
[0040] The light control module is a optogenetic gene expression control module (the light control module is a plasmid or DNA sequence as shown in SEQ ID NO: 1); the CRISPRi module is a dCas9-sgRNA gene inhibition module (the CRISPRi module is a plasmid or DNA sequence as shown in SEQ ID NO: 2);
[0041] The light control module adjusts the expression of the CRISPRi module according to the intensity and time of red and green light irradiation; when the light control module is irradiated with red light, the CRISPRi module is silenced, and the target gene targeted by CRISPRi is normally expressed; when the light control module is irradiated with green light, the CRISPRi module is expressed, and the target gene targeted by CRISPRi is silenced;
[0042] The CRISPRi module can inhibit any target gene in the cell after expression.
[0043] The working principle of the light-controlled CRISPRi gene regulation system is as followsFigure 1 As shown in Figure 1 including Figure 1 -A and Figure 1 -B).
[0044] The principle is: under red light (672nm) irradiation, CcaS and CcaR are not phosphorylated, PcpcG2-172 promoter does not transcribe dCas9 and sgRNA, CRISPRi remains silent, and the target gene can be normally expressed. Under green light (535nm) irradiation, CcaS transfers phosphate group to CcaR, PcpcG2-172 promoter transcribes dCas9 and sgRNA, CRISPRi starts to express, and at this time the target gene is inhibited by CRISPRi and cannot be expressed.
[0045] Example 2, light-controlled CRISPRi gene regulation system inhibits the expression of TetR-eGFP
[0046] 1. Construction of plasmid targeting TetR-eGFP:
[0047] 1.1 PCR replacement sgRNA:
[0048] (1) Design and synthesize the primers in Table 1 according to the sequence information of TetR-eGFP.
[0049] (2) Prepare plasmid template vCQ0, Phusion Flash DNA polymerase, primers, and deionized water, and prepare the PCR reaction system according to Table 2 on ice, mix and centrifuge, and put into the PCR instrument.
[0050] (3) Set the PCR program according to Table 3, and run the program.
[0051] (4) Digest the PCR product with DpnI according to the system in Table 4, and incubate at 37°C for 5 min.
[0052] Table 1: TetR-eGFP primers
[0053]
[0054] Table 2: PCR reaction system
[0055]
[0056]
[0057] Table 3: PCR reaction program
[0058]
[0059] Table 4: DpnI digestion
[0060]
[0061] 1.2 Electrophoresis, gel recovery:
[0062] (1) Take 0.3g agarose, mix with 30ml TAE buffer and heat to melt.
[0063] (2) Add 3μl LabRed nucleic acid dye to the melted gel and mix well.
[0064] (3) Pour the gel solution into the gel holder with inserted combs, and wait for 20min for cooling and solidification.
[0065] (4) Remove the gel combs and load the DpnI digestion product into the sample wells.
[0066] (5) Place the gel containing the sample into the electrophoresis tank, and immerse the gel in TAE buffer.
[0067] (6) Turn on the power of the electrophoresis apparatus, set the voltage to 80V, and run for 45min.
[0068] (7) Take out the gel, and use a scalpel to cut out the target band (5136bp) after taking a picture of the band position using a gel cutter.
[0069] (8) Weigh the gel piece containing the target band, and use a DNA gel recovery kit to purify the fragment.
[0070] 1.3 Transformation of DH5a competent cells:
[0071] (1) Directly add the purified DNA fragment above into a tube of DH5a chemical competent cells, mix gently, and place on ice for 30min.
[0072] (2) Heat shock the competent cells at 42℃ for 45sec, and place on ice for 2min.
[0073] (3) Add 1ml LB liquid medium without antibiotics to the competent cells, and incubate at 37℃ for 1h.
[0074] (4) Pour a piece of LB plate medium containing chloramphenicol.
[0075] (5) Take 100μl of the recovered competent cells and spread on the chloramphenicol plate, and incubate at 37℃ overnight.
[0076] 1.4 Extraction of vCQ1 plasmid:
[0077] (1) Pick a single colony from the plate, inoculate into liquid LB medium containing chloramphenicol, and incubate at 37℃ overnight.
[0078] (2) Use a plasmid DNA extraction kit to extract the vCQ1 plasmid from the overnight culture.
[0079] 2. Integration of light control module and CRISPRi module:
[0080] 2.1 Preparation of MGFROS strain electrocompetent cells:
[0081] (1) Streak the frozen MGFROS strain on a plate containing gentamicin and incubate at 37℃ overnight.
[0082] (2) Pick a single colony and inoculate into liquid LB medium containing gentamicin and incubate at 37℃ overnight.
[0083] (3) Take 1 ml of the overnight culture and add to 100 ml of gentamicin liquid medium and incubate at 37℃ until the OD 600 is 0.5-0.6.
[0084] (4) Divide the bacterial solution into two 50 ml centrifuge tubes and place on ice for 15 min.
[0085] (5) Freeze the cooled bacterial solution at 4℃ and centrifuge at 2500 g for 10 min, discard the medium. Resuspend the bacteria in 25 ml of ice-cold deionized water per tube.
[0086] (6) Freeze at 4℃ and centrifuge at 2500 g for 10 min, discard the supernatant, and resuspend the bacteria in 15 ml of ice-cold 10% glycerol per tube.
[0087] (7) Freeze at 4℃ and centrifuge at 2500 g for 15 min, discard the supernatant, and resuspend the bacteria in ice-cold 10% glycerol again.
[0088] (8) Freeze at 4℃ and centrifuge at 2500 g for 15 min, discard the supernatant, and resuspend the bacteria in 250 μl of ice-cold 10% glycerol per tube, then divide into 1.5 ml centrifuge tubes, 50 μl per tube, and store in a -80℃ freezer for later use.
[0089] 2.2 Electroporation of vCQ1 and vS108 plasmids:
[0090] (1) Take out the frozen electrocompetent cells, vCQ1 and vS108 plasmids.
[0091] (2) Take about 150 ng of each of the two plasmids and mix them well before adding to the electrocompetent cells.
[0092] (3) Add the plasmid-mixed competent cells to the electrotransformation cup and place on ice for 5 min.
[0093] (4) Set the electrotransformation instrument to "bacteria" mode, push the electrotransformation cup into the middle of the electrode plates, and perform a single electroporation with a discharge time of 5-6 ms.
[0094] (5) Immediately add 1 ml of LB liquid medium without antibiotics into the electroporation cup, mix well and then transfer to a 2 ml centrifuge tube, and incubate at 37℃ for 1 h.
[0095] (6) Pour a plate containing chloramphenicol and spectinomycin.
[0096] (7) Take 100 μl of the recovered bacterial solution and spread on a plate containing chloramphenicol and spectinomycin.
[0097] 3. Arabinoe induction experiment:
[0098] 3.1 Culture of bacterial inoculum:
[0099] Pick a single colony and inoculate into double-antibiotic LB liquid medium, and culture overnight in a red light (optimal near 670 nm) irradiation environment.
[0100] 3.2 Preparation of RDM medium containing arabinose:
[0101] (1) In the RDM medium with glycerol as the carbon source, add arabinose as the inducer at a final concentration of 5 mM and 10 mM, respectively.
[0102] (2) Divide a 24-well enzyme plate into 3 groups, and add 1 ml of RDM medium containing 0 mM, 5 mM, and 10 mM arabinose to each well, respectively.
[0103] (3) Inoculate 3 μl of bacterial solution into each well. After completion, seal with parafilm and cover the top with tin paper.
[0104] 3.3 Light-controlled culture and fluorescence detection:
[0105] (1) Place the enzyme plate with the added sample into a light-controlled device that irradiates green light from the bottom, and incubate at 37℃ for 8 h.
[0106] (2) Take out the enzyme plate and place it into an enzyme reader to detect the OD 600 and fluorescence intensity of each well.
[0107] The results are shown in Figure 2 , in the MGFROS strain, TetR-eGFP is inserted downstream of the arabinose promoter, and CRISPRi is designed to target this gene (as shown in Figure 2 -A). When the arabinose concentration in the medium is 5 mM, the inhibition effect of the light-controlled CRISPRi gene regulation system reaches 40 times (as shown in Figure 2 -B).
[0108] Example 3, Light-controlled CRISPRi gene regulation system inhibits the expression of β-galactosidase gene LacZ
[0109] 1. Construction of LacZ plasmid targeting:
[0110] 1.1 PCR replacing sgRNA:
[0111] Design and synthesis of Table 5 primers according to sequence information of LacZ. The rest is the same as Example 2.
[0112] Table 5: LacZ primers
[0113]
[0114] 1.2 Electrophoresis, gel recovery:
[0115] The same as Example 2.
[0116] 1.3 Transformation of DH5a competent cells:
[0117] The same as Example 2.
[0118] 1.4 Extraction of vCQ7 plasmid:
[0119] The same as Example 2.
[0120] 2. Integration of light control module and CRISPRi module:
[0121] 2.1 Preparation of Nissle1917 strain electrotransformation competent cells:
[0122] The rest is the same as Example 2 except that no antibiotics are needed for different strains.
[0123] 2.2 Electroporation co-transformation of vCQ7 and vS108 plasmids:
[0124] The same as Example 2.
[0125] 3. IPTG induction experiment:
[0126] 3.1 Culture of bacterial inoculum:
[0127] Pick a single colony and inoculate it in double-antibiotic liquid LB medium, and culture it in a red light (optimal near 670 nm) irradiation environment to OD 600 0.5-0.6, and obtain bacterial liquid.
[0128] 3.2 Preparation of IPTG plate:
[0129] (1) Pour a piece of LB plate medium containing chloramphenicol and spectinomycin.
[0130] (2) Take 150 μl of mixed solution of X-gal and IPTG, and spread on the surface of the solidified plate medium until completely absorbed.
[0131] 3.3 Light control culture and image acquisition:
[0132] (1) Take 300 μl of the bacterial solution in 3.1 and spread it on the surface of the IPTG plate. Let it stand until it is completely absorbed.
[0133] (2) The plate was irradiated according to the two different strategies (two light control strategies for irradiating X-gal plates: using a tin foil as a mask or using a projector to form a pattern.) as shown in Figure 3 -A. The red and green light was controlled for 12 hours: the tin foil pattern group and the projector group. The tin foil pattern group uses a light control device with a wavelength closer to the absorption peak of the biological element, so the pattern is clearer.
[0134] (3) Take out the light-controlled plate and use a single-lens reflex camera and a film illuminator to collect images, respectively. The results are shown in Figure 3 -B.
[0135] Referring to Figure 3 , after light control by the two strategies, obvious patterns appeared on the X-gal plate coated with the bacterial solution, and the pattern produced by the tin foil pattern group was clearer.
[0136] Example 4, Light-controlled CRISPRi gene regulation system inhibits the expression of FtsZ
[0137] 1. Construction of a plasmid targeting FtsZ:
[0138] 1.1 PCR replacement sgRNA:
[0139] Design and synthesize the primers in Table 6 according to the sequence information of FtsZ. The rest is the same as in Example 2.
[0140] Table 6: FtsZ primers
[0141]
[0142] 1.2 Electrophoresis and gel recovery:
[0143] The same as in Example 2.
[0144] 1.3 Transformation of DH5a competent cells:
[0145] The same as in Example 2.
[0146] 1.4 Extraction of vCQ5 plasmid:
[0147] The same as in Example 2.
[0148] 2. Integration of light control module and CRISPRi module:
[0149] 2.1 Preparation of MGFROS bacterial strain electroporation competent cells:
[0150] The same as in Example 2.
[0151] 2.2 Electroporation of vCQ5 and vS108 plasmids:
[0152] Same as Example 2.
[0153] 3. Microfluidic observation experiment:
[0154] 3.1 Culture of bacterial inoculum:
[0155] A single colony was picked and inoculated into 15 ml of LB medium with antibiotics and cultured for 5 h in a red light (optimal near 670 nm) irradiation environment.
[0156] 3.2 Sample loading on microfluidic chip:
[0157] (1) Use a sterile syringe to push the sterile culture medium containing surfactant into the capillary and the interior of the microfluidic chip to remove air bubbles.
[0158] (2) Centrifuge the above bacterial solution at 12°C to concentrate it to about 0.5 ml.
[0159] (3) Push the concentrated solution into the channel of the microfluidic chip.
[0160] 3.3 Light-controlled culture and microscopic imaging:
[0161] (1) Install the syringe containing the double-antibiotic culture medium (containing surfactant) in the peristaltic pump and set it to push the culture medium into the chip at a constant speed.
[0162] (2) Fix the chip in the incubator and set it to a constant temperature of 37°C.
[0163] (3) Place the incubator on the stage of the Nikon Ti2 inverted microscope and install an external annular green light source between the bright field light source and the chip, continuously irradiating the bacteria in the chip for 4 h.
[0164] (4) Focus on the bacteria in the chip and set the microscope to automatically capture a bright field image every 30 seconds. Reference Figure 4 , the results show that after the bacteria are transferred from red light culture to green light culture, they cannot divide due to the inhibition of FtsZ by CRISPRi, forming typical filamentous cells.
[0165] Example 5, Light-controlled CRISPRi gene regulation system inhibits the expression of MreB
[0166] 1. Construction of MreB-targeting plasmid:
[0167] 1.1 PCR replacement of sgRNA:
[0168] Design and synthesize primers according to the sequence information of MreB. The rest is the same as Example 2.
[0169] Table 7: MreB primers
[0170]
[0171] 1.2 Electrophoresis, gel recovery:
[0172] The same as in Example 2.
[0173] 1.3 Transformation of DH5a competence:
[0174] The same as in Example 2.
[0175] 1.4 Extraction of vCQ6 plasmid:
[0176] The same as in Example 2.
[0177] 2. Integration of light control module and CRISPRi module:
[0178] 2.1 Preparation of MGFROS strain electroporation competence:
[0179] The same as in Example 2.
[0180] 2.2 Electroporation co-transformation of vCQ6 and vS108 plasmids:
[0181] The same as in Example 2.
[0182] 3. Microfluidic observation experiment:
[0183] 3.1 Culture of bacterial inoculum:
[0184] The same as in Example 4.
[0185] 3.2 Microfluidic chip loading:
[0186] The same as in Example 4.
[0187] 3.3 Light control culture and microscopic imaging:
[0188] The same as in Example 4.
[0189] Reference Figure 5 Figure 6 shows that after the bacteria are switched from red light culture to green light culture, MreB cannot maintain short rod shape due to inhibition by CRISPRi, and typical round cells are formed.
[0190] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.
Claims
1. A light-controlled CRISPRi gene regulation system, characterized in that, The light-controlled CRISPRi gene regulation system comprises a biological circuit and a light source device; the biological circuit comprises a light-controlled module and a CRISPRi module; the light source device is an electronic device emitting red light or green light; The light control module adjusts the expression of the CRISPRi module according to the intensity and time of red and green light irradiation; when the light control module is irradiated with red light, the CRISPRi module is silenced, and the target gene targeted by the CRISPRi is normally expressed; when the light control module is irradiated with green light, the CRISPRi module is expressed, and the target gene targeted by the CRISPRi is silenced; the light control module comprises PcpcG2 172 promoter and RBS; The CRISPRi module can inhibit any target gene in a cell after expression; The light-controlled module is a plasmid or DNA sequence as shown in SEQ NO: 1; and the CRISPRi module is a plasmid or DNA sequence as shown in SEQ NO:
2.
2. The light-controlled CRISPRi gene regulation system of claim 1, wherein, The CRISPRi module is a dCas9-sgRNA gene inhibition module.
3. The light-controlled CRISPRi gene regulation system of claim 1, wherein, The target gene of the light-controlled CRISPRi gene regulation system can be changed by only changing the target sequence of sgRNA of the CRISPRi module.
4. The light-controlled CRISPRi gene regulation system of claim 1, wherein, The emission wavelengths of red light and green light are wavelengths capable of causing a photo-genetic molecular response.
5. A method of constructing a light-controlled CRISPRi gene regulation system according to any one of claims 1-4, characterized in that, The method comprises the following steps: Integrating related genes on a biological circuit, wherein the biological circuit comprises a light-controlled module and a CRISPRi module; integrating the light-controlled module and the CRISPRi module into a cell; simultaneously constructing a light source device to form a light-controlled CRISPRi gene regulation system.
6. Use of the light-controlled CRISPRi gene regulation system according to any one of claims 1-4 to turn on or turn off the expression of a target gene in a host cell.