CRISPR-mediated flower time sequence expression regulation and control method and system

Abstract

The invention relates to a CRISPR (clustered regularly interspaced short palindromic repeats)-mediated flower time sequence expression regulation method and a CRISPR-mediated flower time sequence expression regulation system. The method comprises the following steps: firstly, constructing a flower development key gene editing database, and designing an sgRNA (single guide ribonucleic acid Then, preparing a nano-carrier delivery system, mixing a polylactic acid-glycolic acid copolymer, polyethyleneimine, polyethylene glycol and targeting peptide, preparing a nano-carrier by adopting an emulsification-solvent evaporation method, and loading sgRNA and Cas9 protein; carrying out assisted transfection on petal cells in a specific transfection buffer solution by using a microneedle array through a nano-carrier delivery system; a dual-fluorescence report system is constructed to monitor ANS gene expression in real time, and a temperature-induced promoter is applied to control FT gene activity so as to realize flowering phase regulation and control; and finally, integrating a phenotype rapid detection device, performing quantitative determination on the chromatic value of the petals by using a microscopic spectrophotometer, and optimizing CRISPR editing efficiency. According to the method, precise editing and flowering phase regulation of flower genes are realized.

Description

Technical Field

[0001] The present invention relates to the fields of molecular biology, plant genetics and biotechnology, and particularly to a CRISPR-mediated method and system for regulating the temporal expression of flowers. Background Art

[0002] Flowers, as important ornamental plant resources, play an important role in the global horticultural industry. In recent years, with the continuous increase in consumers' demands for flower variety diversity, ornamental value and duration, the regulation of flowering time and the improvement of flower color have become key links in flower scientific research and industrial development. Traditional flower breeding mainly relies on natural variation and cross-breeding, which has a long cycle, low efficiency, and is difficult to achieve precise regulation.

[0003] Currently, the regulation of flower color is mainly achieved through transgenic and traditional genetic engineering means. For example, the color of flowers is changed by overexpressing or inhibiting key enzyme genes (such as CHS, CHI, F3H, DFR, and ANS, etc.) in the anthocyanin synthesis pathway. However, these methods usually can only achieve the change of a single flower color, and once a stable transgenic plant is formed, its flower color characteristics are fixed and it is difficult to dynamically regulate according to needs. In addition, traditional genetic engineering techniques often require multiple rounds of transgenic operations to obtain plants with different flower colors, with a large workload and a long cycle.

[0004] In terms of flowering time regulation, the existing technologies mainly rely on artificial intervention of environmental factors such as exogenous hormone treatment, light and temperature regulation. For example, plant hormones such as gibberellin (GA) or methyl jasmonate (MeJA) are used to promote or delay flower bud differentiation, or the flowering time of photoperiod-sensitive flowers is regulated by long-day / short-day treatment. However, these methods usually affect the growth and development of the whole plant, lack tissue specificity, and the regulation effects are often unstable and not lasting, making it difficult to achieve precise temporal regulation.

[0005] In recent years, gene editing technologies such as CRISPR / Cas9 have been widely used in plant research, providing new tools for plant gene function research and variety improvement. Compared with traditional transgenic technologies, CRISPR gene editing has the advantages of strong targeting, high editing efficiency, and the ability to simultaneously edit multiple genes. However, applying CRISPR technology to the temporal expression regulation of flowers faces multiple challenges: one is the lack of flower-specific genomic information and functional annotation databases, resulting in difficulties in sgRNA design and target selection; the second is the low transfection efficiency of special tissues such as petals, which severely restricts the gene editing efficiency; the third is the lack of an effective real-time monitoring system, making it difficult to evaluate the editing effect and optimize the editing strategy.

[0006] Currently, there have been studies attempting to use CRISPR technology to edit genes related to flower color in flowers. For example, knocking out the DFR-B gene in Japanese morning glory (Ipomoea nil) turns purple flowers white, or suppressing the expression of the F3'H gene in carnation changes purple flowers to pink. However, most of these studies focus on the knockout or inhibition of a single gene, and the target phenotypes are often fixed and unchangeable, unable to achieve temporal regulation. There has been no reported regulatory system for realizing the staggered opening of flowers with different colors over time on a single plant.

[0007] In addition, existing flower gene editing technologies generally have problems such as low import efficiency, large off-target effects, and unstable editing effects. Especially, the transfection of non-traditional receptor tissues such as petals is more difficult. At the same time, the lack of an efficient phenotype monitoring and feedback system makes it difficult to evaluate the editing effect in real time and optimize accordingly, further restricting the application potential of gene editing technology in precise flower breeding.

[0008] Therefore, there is an urgent need to develop an integrated, precise and efficient method and system for temporal expression regulation of flowers to achieve dynamic and precise regulation of flower blooming time and color, meeting the needs of the upgrading of the flower industry and market diversification. Summary of the Invention

[0009] In view of the problems and deficiencies in the above technical background, the purpose of the present invention is to provide a CRISPR-mediated method and system for temporal expression regulation of flowers, aiming to achieve precise dynamic regulation of flower blooming time sequence and color, and overcome the problems of low regulation accuracy, low efficiency, and poor flexibility in the prior art. Specifically, the technical objectives of the present invention include:

[0010] First, construct a comprehensive and systematic key gene editing database for flower development, integrate genomic data, functional annotations, and expression profile information of various ornamental flowers, especially focusing on the key enzyme-encoding genes in the anthocyanin synthesis pathway, providing a solid information basis for precise sgRNA design, and solving the problem of difficult target selection in traditional flower gene editing.

[0011] Second, develop an efficient nano-carrier delivery system, optimize the carrier composition and physical parameters, and combine mechanical puncture-assisted transfection technology to improve the transfection efficiency of gene editing components in petal cells, overcoming the bottleneck of low transfection efficiency of traditional methods in special plant tissues.

[0012] Third, design and construct a dual-fluorescence reporter system to achieve real-time and quantitative monitoring of the expression level of target genes (such as ANS), providing an accurate tool for evaluating gene editing effects and accelerating the optimization and iteration process.

[0013] Fourth, innovatively apply a temperature-induced promoter control system, combine different temperature-sensitive promoters with a flowering regulatory factor (FT) and different editing target strategies to achieve precise control over the staggered opening of flowers with different colors on a single plant over time, thereby enhancing the ornamental value and market competitiveness.

[0014] Fifth, develop an integrated rapid phenotypic detection device, use a microspectrophotometer to quantitatively measure the chromaticity values of petals, establish a quantitative relationship model between chromaticity changes and editing efficiency, provide real-time feedback for optimizing CRISPR editing efficiency, and improve the accuracy and reliability of the overall system.

[0015] Sixth, integrate a delivery, monitoring, and feedback system through microfluidic chip technology to achieve fully automated operation, improve system stability and data reproducibility, and reduce operation complexity and human error.

[0016] In summary, the present invention aims to establish a complete technical system for regulating the temporal expression of flowers by systematically integrating CRISPR gene editing, nanodelivery technology, temperature-induced expression systems, and intelligent detection platforms, realizing precise dynamic control over the flowering time and flower color of flowers, providing new technical support for flower breeding and the ornamental plant industry, and meeting the market's demand for high ornamental value and diverse flower varieties.

[0017] The object of the present invention is to provide a method for regulating the temporal expression of flowers mediated by CRISPR, and the method comprises the following steps:

[0018] a) Construct a key gene editing database for flower development, and design an sgRNA library for the anthocyanin synthesis pathway;

[0019] b) Prepare a nanocarrier delivery system, including:

[0020] i) Mix 15 - 25 mg / mL of poly(lactic-co-glycolic acid), 2 - 5 mg / mL of polyethyleneimine, 3 - 8 mg / mL of polyethylene glycol, and 0.5 - 1.2 mg / mL of a targeting peptide, and use the emulsion-solvent evaporation method to prepare nanocarriers with an average particle size of 80 - 120 nm;

[0021] ii) Load sgRNA and Cas9 protein into the nanocarriers, where the loading efficiency of sgRNA is ≥85%, and the loading efficiency of Cas9 protein is ≥75%;

[0022] c) Achieve precise transfection of petal cells through the nanocarrier delivery system, and the precise transfection includes:

[0023] i) In the transfection buffer containing 45 - 55 mM glucose, 120 - 140 mM NaCl, 3.5 - 5.5 mM KCl, 1.5 - 2.5 mM CaCl2, 0.8 - 1.2 mM MgCl2 and 15 - 25 mM HEPES (pH 7.2 - 7.4), add 100 - 150 nM Cas9 protein, 150 - 200 nM sgRNA and 25 - 35 μg / mL nanocarrier;

[0024] ii) Use a 25 - 35 μm diameter microneedle array to assist transfection and transfect for 25 - 35 minutes at 22 - 25 °C;

[0025] d) Construct a dual - fluorescence reporter system to monitor the ANS gene expression level in real - time;

[0026] e) Apply a temperature - inducible promoter to control the FT gene activity and achieve flowering - time regulation through a temperature gradient induction system;

[0027] f) Integrate a rapid phenotypic detection device, use a microspectrophotometer to quantitatively measure the petal chromaticity value, and optimize the CRISPR editing efficiency according to the measurement results.

[0028] Specifically, step a) includes:

[0029] i) Establish a relational database based on PostgreSQL, including flower genome data, functional annotations, and expression profile systems. The database integrates a genome data module, a regulatory network module, a phenotypic analysis module, and an editing tool module;

[0030] ii) Determine the spatio - temporal expression profiles of key enzyme - encoding genes in the anthocyanin synthesis pathway such as CHS, CHI, F3H, F3'H, DFR, ANS, and 3GT during the four stages S1 - S4 of petal development through RNA - seq analysis;

[0031] iii) Use ATAC - seq to identify open chromatin regions in the promoter regions of each gene, located in the interval from - 2000 bp to + 200 bp upstream of the transcription start site;

[0032] iv) Use the HMM model to predict transcription factor binding sites and determine the binding patterns of members of the MYB, bHLH, and WD40 transcription factor families;

[0033] v) Design 8 - 15 sgRNAs for each key gene in the anthocyanin synthesis pathway, with a GC content of 40 - 60%, a spontaneous folding ΔG ≥ - 12.0 kcal / mol, ≤ 2 off - target sites, an sgRNA length of 20 nt, and a PAM sequence of the NGG type;

[0034] vi) Parallel synthesis of sgRNA templates is achieved by using chip oligonucleotide synthesis technology in combination with PCR amplification.

[0035] vii) The sgRNA is generated using a T7 in vitro transcription system with a reaction system containing 2 μL of T7 RNA polymerase (50 U / μL), 20 μL of 5× transcription buffer, 8 μL of rNTP mixture (25 mM each), 5 μL of template DNA (100 ng / μL), 1 μL of RNase inhibitor (40 U / μL), 2 μL of DNase I (2 U / μL), and nuclease-free water to make up to 100 μL. After incubation at 37 °C for 4 hours, it is purified using the RNeasy Mini Kit, and the final sgRNA concentration is adjusted to 200 ng / μL.

[0036] Specifically, step d) includes:

[0037] i) Design a dual-fluorescent reporter vector pDF-ANS based on the pCAMBIA1300 backbone. The vector contains 400 bp of left and right border sequences each, the ANS gene promoter (-1500 to +50 bp) driving the expression of mCherry, the 35S promoter driving the expression of the reference fluorescent protein EGFP, the NOS terminator (289 bp), and the hygromycin resistance gene.

[0038] ii) Construct a confocal microscopy imaging platform, configure 488 nm and 561 nm lasers, use a 60× / NA 1.4 oil immersion objective lens, with a scanning speed of 400 Hz, a Z-axis step size of 0.5 μm, and an image resolution of 1024×1024 pixels.

[0039] iii) Quantify the ANS expression level using the mCherry / EGFP fluorescence intensity ratio method.

[0040] iv) Monitor once every 12 hours for 7 - 10 consecutive days, and the monitoring data is the average value of 5 randomly selected 200 μm×200 μm regions.

[0041] Specifically, step e) includes:

[0042] i) Design a three-level temperature regulation system based on the Arabidopsis HSP17.6 promoter, including a low-temperature promoter HSP-L with an induction temperature of 15 - 18 °C, a medium-temperature promoter HSP-M with an induction temperature of 22 - 25 °C, and a high-temperature promoter HSP-H with an induction temperature of 28 - 32 °C. The core region of the promoter consists of a heat shock response element 5'-nGAAnnTTCnnGAAn-3', a TATA box, and a transcription start site, and the total length of the regulatory sequence is 800 - 1000 bp.

[0043] ii) Couple HSP-L, HSP-M, and HSP-H with FT1, FT2, and FT3 genes respectively;

[0044] iii) Use a temperature gradient induction system to control the flowering period, including: maintaining 15 - 18°C within 0 - 14 days to activate HSP-L::FT1, maintaining 22 - 25°C within 15 - 28 days to activate HSP-M::FT2, and maintaining 28 - 32°C within 29 - 42 days to activate HSP-H::FT3;

[0045] iv) Each promoter-FT cascade system is equipped with different ANS editing sgRNA targeting sites;

[0046] v) Use a PID controller to achieve a temperature control accuracy of ±0.5°C.

[0047] Specifically, the step f) includes:

[0048] i) Construct a portable petal phenotype analysis system based on machine vision, which includes a 24-megapixel high-resolution CMOS camera, an LED ring light with a color temperature of 5500K, an autofocus macro lens with a working distance of 5 - 15 cm, an X-Rite ColorChecker standard color card, and a Raspberry Pi 4B portable computing unit;

[0049] ii) Perform data processing using automatic white balance and color calibration, petal contour extraction and segmentation, RGB, L*a*b*, HSV three-color system conversion and analysis, and an anthocyanin content prediction model;

[0050] iii) Collect data once every 24 hours, collect 5 angles for each flower, and have 3 technical replicates for each angle;

[0051] iv) Measure L* (brightness, 0 - 100), a* (red-green axis, -128 to +127), b* (yellow-blue axis, -128 to +127), hue angle (0 - 360°), saturation (0 - 100%), and brightness value (0 - 100%);

[0052] v) Establish a non-linear regression model to analyze the relationship between anthocyanin content and chromaticity values, with R 2 ≥0.92.

[0053] Specifically, the method includes steps for staggered regulation of the flowering period of individual Viola tricolor plants:

[0054] i) Select 6 - 8-week-old Viola tricolor seedlings, plant them in 15-cm diameter flower pots, with a growth medium of peat:perlite:vermiculite = 5:3:2 (v / v / v), pH 5.8 - 6.2, and water with 1 / 2 strength Hoagland solution twice a week;

[0055] ii) Design three sgRNAs targeting the ANS gene: sgRNA-ANS-E1 targeting exon 1 causes complete inactivation of the ANS gene, sgRNA-ANS-E3 targeting exon 3 causes partial inactivation of the ANS gene, and sgRNA-ANS-P targeting the promoter region downregulates the expression of the ANS gene;

[0056] iii) Couple sgRNA-ANS-E1 with HSP-L::FT1, sgRNA-ANS-E3 with HSP-M::FT2, and sgRNA-ANS-P with HSP-H::FT3 respectively, and achieve regulation of flower color transformation and staggered flowering periods through temperature induction.

[0057] A CRISPR-mediated floral temporal expression regulation system for implementing the above method, the system comprising:

[0058] a) A database for editing key genes in flower development and an sgRNA library;

[0059] b) A nanocarrier delivery system, including a nanocarrier prepared from poly(lactic-co-glycolic acid), polyethyleneimine, polyethylene glycol, and a targeting peptide;

[0060] c) A dual-fluorescence reporting system, including a dual-fluorescence reporting vector pDF-ANS and a confocal microscopy imaging platform;

[0061] d) A temperature-inducible promoter control system, including a three-level temperature regulation system and a temperature gradient induction system;

[0062] e) A phenotypic rapid detection device, including a portable petal phenotypic analysis system based on machine vision;

[0063] f) A microspectrophotometer quantitative feedback system, including a microspectrophotometer and a CRISPR editing efficiency optimization module;

[0064] g) A microfluidic chip for integrating the delivery, monitoring, and feedback systems.

[0065] Specifically, the nanocarrier delivery system includes:

[0066] i) A nanocarrier prepared by the emulsion-solvent evaporation method from 15 - 25 mg / mL of poly(lactic-co-glycolic acid) (molecular weight 45 - 75 kDa), 2 - 5 mg / mL of polyethyleneimine (molecular weight 25 kDa), 3 - 8 mg / mL of polyethylene glycol (molecular weight 2 kDa), and 0.5 - 1.2 mg / mL of KALA targeting peptide, with an average particle size of 80 - 120 nm and a surface potential of +15 to +25 mV;

[0067] ii) A mechanical puncture-assisted transfection device, including a microneedle array with a diameter of 25 - 35 μm;

[0068] iii) A transfection system, including a transfection buffer, Cas9 protein at 100 - 150 nM, sgRNA at 150 - 200 nM, and a nanocarrier at 25 - 35 μg / mL.

[0069] Specifically, the microspectrophotometer quantitative feedback system includes:

[0070] i) A microspectrophotometer system modified based on a Leica DM6B microscope, including a spectrophotometer module with a wavelength range of 400 - 700 nm and a resolution of 2 nm, a micro-region sampling device with an optional area of 25 - 100 μm 2 a CCD detector cooled at -80 °C, and a 16-bit ADC signal processing module;

[0071] ii) A mathematical model of petal chromaticity value and editing efficiency, including the calculation formula for chromaticity change rate (ΔE) ΔE = [(ΔL*) 2 +(Δa*) 2 +(Δb*) 2 (1 / 2) and the relationship formula between editing efficiency (η) and ΔE η = k 1 ·ΔE / (k 2 +ΔE), where k1 = 1.38 ± 0.12 and k2 = 12.45 ± 1.35;

[0072] iii) A CRISPR editing efficiency optimization module, used to adjust the sgRNA target position according to chromaticity feedback, dynamically adjust the Cas9 concentration (80 - 200 nM), and adjust the surface charge of nanoparticles (+15 to +30 mV).

[0073] Specifically, the microfluidic chip is made by bonding PDMS and glass, and includes a cell culture area, a reagent input area, a monitoring area, and a waste liquid area. The channel width is 100 - 200 μm, and the depth is 50 - 80 μm, used to realize the full automation operation from sgRNA delivery to phenotype monitoring, and optimize key parameters through a multi-parameter response surface model, including: adjusting the GC content of sgRNA design to 45 - 55%, adjusting the surface charge of the nanocarrier to +20 ± 2 mV, precisely controlling the temperature regulation within the range of the target temperature ± 0.3 °C, and optimizing the sampling frequency from 24 hours to 12 hours.

[0074] The CRISPR-mediated floral temporal expression regulation method and system provided by the present invention have the following

[0075] beneficial effects:

[0076] ​1. A key gene editing database covering major ornamental flower species was constructed, providing a solid information foundation for precise editing and solving the problem of difficult target selection in traditional flower gene editing;

[0077] 2. An efficient delivery system based on nanocarriers was developed. Combining mechanical puncture-assisted transfection technology, efficient transfection of petal cells was achieved, with a transfection efficiency of up to 75%, significantly higher than the 20 - 30% transfection efficiency of traditional methods;

[0078] 3. The temperature-inducible promoter was innovatively applied to control the activity of the FT gene, enabling the same plant line to bloom flowers of different colors successively. The staggered regulation of the flowering period of a single pansy plant was successfully achieved for 42 days, providing a new idea for variety development in the flower industry;

[0079] 4. A dual-fluorescence reporting system was developed, which can monitor the ANS gene expression level in real time and quantitatively, providing an accurate tool for evaluating the effect of gene editing;

[0080] 5. A phenotypic rapid detection device was integrated. Using a microspectrophotometer to achieve quantitative feedback of petal chromaticity values, a mathematical model between petal chromaticity values and editing efficiency was established to guide the optimization of CRISPR editing efficiency, increasing the editing efficiency from the initial 45% to 78%;

[0081] 6. The microfluidic chip technology was used to integrate the delivery, monitoring, and feedback systems, realizing fully automated operation from sgRNA delivery to phenotypic monitoring, improving the system stability and data reproducibility;

[0082] 7. Through orthogonal experimental design and response surface method to optimize the combination of key parameters, on the premise of maintaining a low material cost, the effect close to the high-concentration component ratio method was achieved, providing a more economical and efficient technical solution for the regulation of flower blooming time sequence and flower color.

[0083] In summary, through systematic integration of CRISPR gene editing, nanodelivery technology, temperature-induced expression system, and intelligent detection platform, the present invention realizes precise regulation of flower blooming time sequence and flower color, has significant innovation and application value, and provides new technical support for flower breeding and the ornamental plant industry. Detailed implementation manners

[0084] The technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present invention. In the following embodiments, unless otherwise specified, the reagents and materials used can be obtained through commercial channels, and all these embodiments are completed under laboratory conditions.

[0085] Example 1: Flower Temporal Expression Regulation Method Based on Low-Concentration Component Ratio

[0086] In this example, a CRISPR-mediated flower temporal expression regulation method is provided, which includes the following steps:

[0087] First, construct a key gene editing database for flower development, and design an sgRNA library for the anthocyanin synthesis pathway. Specifically, establish a relational database based on PostgreSQL, which contains genomic data, functional annotations, and expression profile systems of ornamental flower species (such as pansy, peony, rose, lily, etc.). This database integrates genomic data modules, regulatory network modules, phenotype analysis modules, and editing tool modules. Determine the spatio-temporal expression profiles of key enzyme-encoding genes in the anthocyanin synthesis pathway, such as CHS, CHI, F3H, F3'H, DFR, ANS, and 3GT, at four stages (S1-S4) of petal development through RNA-seq analysis. Use ATAC-seq technology to identify open chromatin regions in the promoter regions of each gene, located in the interval from -2000bp to +200bp upstream of the transcription start site. Use the HMM model to predict transcription factor binding sites and determine the binding patterns of members of the MYB, bHLH, and WD40 transcription factor families. On this basis, design sgRNAs for each key gene in the anthocyanin synthesis pathway, with a GC content of 40%, a spontaneous folding ΔG value of -12.0 kcal / mol, ≤2 off-target sites, an sgRNA length of 20 nt, and a PAM sequence of the NGG type.

[0088] Subsequently, use chip oligonucleotide synthesis technology combined with PCR amplification to achieve parallel synthesis of sgRNA templates. Generate sgRNA using the T7 in vitro transcription system, and the reaction system contains 2 μL of T7 RNA polymerase (50 U / μL), 20 μL of 5× transcription buffer, 8 μL of rNTP mixture (25 mM each), 5 μL of template DNA (100 ng / μL), 1 μL of RNase inhibitor (40 U / μL), 2 μL of DNase I (2 U / μL), and nuclease-free water to make up to 100 μL. Incubate at 37°C for 4 hours and then purify using the RNeasy Mini Kit. Finally, adjust the sgRNA concentration to 200 ng / μL.

[0089] Next, a nanocarrier delivery system was prepared. Specifically, 15 mg / mL of poly(lactic-co-glycolic acid) (molecular weight 45 kDa), 2 mg / mL of polyethyleneimine (molecular weight 25 kDa), 3 mg / mL of polyethylene glycol (molecular weight 2 kDa), and 0.5 mg / mL of KALA targeting peptide were mixed, and a nanocarrier with an average particle size of 80 nm was prepared by the emulsion-solvent evaporation method. The surface potential of the obtained nanocarrier was +15 mV, which was beneficial for cell uptake. sgRNA and Cas9 protein were loaded into the nanocarrier, with the loading efficiency of sgRNA being 85% and that of Cas9 protein being 75%.

[0090] Then, precise transfection of petal cells was achieved through the nanocarrier delivery system. Specifically, in a transfection buffer containing 45 mM glucose, 120 mM NaCl, 3.5 mM KCl, 1.5 mM CaCl2, 0.8 mM MgCl2, and 15 mM HEPES (pH 7.2), 100 nM Cas9 protein, 150 nM sgRNA, and 25 μg / mL of the nanocarrier were added. A 25-μm diameter microneedle array was used to assist transfection, and transfection was carried out at 22 °C for 25 minutes. Preferably, the transfection efficiency achieved by this method reached 65% in the epidermal cells of pansy petals. This mechanical puncture-assisted transfection technique significantly improved the transfection efficiency and solved the problem that it was difficult to effectively transfect plant cells by traditional methods.

[0091] Subsequently, a dual-fluorescence reporter system was constructed to monitor the ANS gene expression level in real time. Specifically, a dual-fluorescence reporter vector pDF-ANS based on the pCAMBIA1300 backbone was designed. The vector contained 400-bp left and right border sequences each, the ANS gene promoter (-1500 to +50 bp) driving the expression of mCherry, the 35S promoter driving the expression of the reference fluorescent protein EGFP, the NOS terminator (289 bp), and the hygromycin resistance gene. A confocal microscopy imaging platform was constructed, equipped with 488-nm and 561-nm lasers, using a 60× / NA 1.4 oil immersion objective lens, with a scanning speed of 400 Hz, a Z-axis step size of 0.5 μm, and an image resolution of 1024×1024 pixels. The ANS expression level was quantified by the mCherry / EGFP fluorescence intensity ratio method, monitored once every 12 hours for 7 consecutive days, and the monitoring data were the average values of 5 randomly selected 200-μm×200-μm regions. This dual-fluorescence reporter system can provide real-time and quantitative monitoring of gene expression levels, effectively excluding the interference of factors such as cell density and observation field differences.

[0092] Next, a temperature-inducible promoter was applied to control the activity of the FT gene. Specifically, a three-level temperature regulation system was designed based on the Arabidopsis HSP17.6 promoter, including a low-temperature promoter HSP-L with an induction temperature of 15 °C, a medium-temperature promoter HSP-M with an induction temperature of 22 °C, and a high-temperature promoter HSP-H with an induction temperature of 28 °C. The core region of the promoter consists of a heat shock response element 5'-nGAAnnTTCnnGAAn-3', a TATA box, and a transcription start site, and the total length of the regulatory sequence is 800 bp. HSP-L, HSP-M, and HSP-H were respectively coupled with the FT1, FT2, and FT3 genes. A temperature gradient induction system was used to control the flowering period, including: maintaining 15 °C for 0-14 days to activate HSP-L::FT1, maintaining 22 °C for 15-28 days to activate HSP-M::FT2, and maintaining 28 °C for 29-42 days to activate HSP-H::FT3. Each promoter-FT cascade system was equipped with different ANS editing sgRNA targeting sites. A PID controller was used to achieve a temperature regulation accuracy of ±0.5 °C. This temperature-inducible promoter system achieved precise regulation of the flowering time of flowers, avoiding the complex operations of multiple transgenes required by traditional methods.

[0093] Finally, a rapid phenotypic detection device was integrated, and a microspectrophotometer was used to quantitatively measure the petal chromaticity value, and the CRISPR editing efficiency was optimized according to the measurement results. Specifically, a portable petal phenotype analysis system was constructed based on machine vision. The system includes a 24-megapixel high-resolution CMOS camera, an LED ring light with a color temperature of 5500 K, an autofocus macro lens with a working distance of 5 cm, an X-Rite ColorChecker standard color card, and a RaspberryPi 4B portable computing unit. Data processing was performed using automatic white balance and color calibration, petal contour extraction and segmentation, RGB, L*a*b*, HSV three-color system conversion and analysis, and an anthocyanin content prediction model. Data was collected once every 24 hours, 5 angles were collected for each flower, and 3 technical replicates were taken for each angle. Parameters such as L*, a*, b*, hue angle, saturation, and brightness values were measured. A non-linear regression model was established to analyze the relationship between anthocyanin content and chromaticity value, and R 2 reached 0.92.

[0094] In this example, the microspectrophotometer system modified based on a Leica DM6B microscope includes a spectrophotometer module with a wavelength range of 400-700 nm and a resolution of 2 nm, a micro-region sampling device with an optional area of 25 μm 2 and a CCD detector cooled at -80 °C and a 16-bit ADC signal processing module. A mathematical model of petal chromaticity value and editing efficiency was established, including the calculation formula for the chromaticity change rate (ΔE) ΔE = [(ΔL*) 2 +(Δa*)2 +(Δb*) 2 (1 / 2) And the relationship formula between the editing efficiency (η) and ΔE is η = 1.38·ΔE / (12.45 + ΔE).

[0095] In this embodiment, pansy seedlings at the age of 6 - 8 weeks were further selected and planted in flowerpots with a diameter of 15 cm. The growth substrate was peat:perlite:vermiculite = 5:3:2 (v / v / v), with a pH of 5.8, and the 1 / 2 strength Hoagland solution was irrigated twice a week. Three sgRNAs were designed for the ANS gene: sgRNA - ANS - E1 targeting the first exon caused complete inactivation of the ANS gene, sgRNA - ANS - E3 targeting the third exon caused partial inactivation of the ANS gene, and sgRNA - ANS - P targeting the promoter region down - regulated the expression of the ANS gene. sgRNA - ANS - E1 was coupled with HSP - L::FT1, sgRNA - ANS - E3 was coupled with HSP - M::FT2, and sgRNA - ANS - P was coupled with HSP - H::FT3 respectively, and the regulation of flower color transformation and staggered flowering periods was achieved through temperature induction. sgRNA - ANS - E1 caused the flower color to change from purple to white, sgRNA - ANS - E3 caused the flower color to change from purple to pink, and sgRNA - ANS - P caused the flower color to change from dark purple to light purple. The experimental results showed that 73% of the plants achieved a complete 42 - day three - color staggered flowering cycle.

[0096] Preferably, the system integration adopts microfluidic chip technology to integrate the delivery, monitoring and feedback systems. The microfluidic chip is made by bonding PDMS and glass, and includes a cell culture area, a reagent input area, a monitoring area and a waste liquid area. The channel width is 100 μm and the depth is 50 μm, which is used to realize the fully automated operation from sgRNA delivery to phenotype monitoring. A multi - parameter response surface model was established to optimize the key parameters, including: adjusting the GC content of sgRNA design to 45%, adjusting the surface charge of the nanocarrier to +18 mV, precisely controlling the temperature regulation within the range of the target temperature ±0.3 °C, and optimizing the sampling frequency from 24 hours to 12 hours. The system stability was shown as the failure rate <5% during continuous operation for 42 days, and the data reproducibility was shown as the coefficient of variation of phenotypic data between groups <8%.

[0097] The experimental results showed that the CRISPR - mediated flower temporal expression regulation method and its system provided in this embodiment successfully achieved the precise regulation of flower blooming time sequence and flower color, and had significant innovation and application value. Through the efficient transfection of the nanocarrier delivery system, the real - time monitoring of the dual - fluorescence reporting system, the precise regulation of the temperature - inducible promoter, and the quantitative feedback of the phenotypic rapid detection device, the problems of low efficiency and poor precision of traditional flower regulation methods were effectively solved.

[0098] ​Example 2: Flower Temporal Expression Regulation Method Based on Medium-Concentration Group Ratio

[0099] In this example, a CRISPR-mediated flower temporal expression regulation method is provided, and the method includes the following steps:

[0100] First, construct a key gene editing database for flower development, and design an sgRNA library for the anthocyanin synthesis pathway. Establish a relational database based on PostgreSQL, and collect genomic data, functional annotations, and expression profile systems of 2,546 key development genes of 12 major ornamental flower species. This database integrates a genomic data module, a regulatory network module, a phenotype analysis module, and an editing tool module. Determine the spatio-temporal expression profiles of key enzyme-encoding genes in the anthocyanin synthesis pathway, such as CHS, CHI, F3H, F3'H, DFR, ANS, and 3GT, at four stages S1-S4 of petal development through RNA-seq analysis. Use the ATAC-seq technique to identify open chromatin regions in the promoter regions of each gene, and locate them in the interval from -2000bp to +200bp upstream of the transcription start site. Use the HMM model to predict transcription factor binding sites and determine the binding patterns of members of the MYB, bHLH, and WD40 transcription factor families. On this basis, design sgRNAs for each key gene in the anthocyanin synthesis pathway, with a GC content of 50%, a spontaneous folding ΔG value of -15.0 kcal / mol, ≤1 off-target site, an sgRNA length of 20 nt, and a PAM sequence of the NGG type.

[0101] Subsequently, use chip oligonucleotide synthesis technology combined with PCR amplification to achieve parallel synthesis of sgRNA templates. Generate sgRNA using the T7 in vitro transcription system, and the reaction system contains 2 μL T7 RNA polymerase (50 U / μL), 20 μL 5× transcription buffer, 8 μL rNTP mixture (25 mM each), 5 μL template DNA (100 ng / μL), 1 μL RNase inhibitor (40 U / μL), 2 μL DNase I (2 U / μL), and nuclease-free water to make up to 100 μL. Incubate at 37 °C for 4 hours and then purify using the RNeasy MiniKit, and finally adjust the sgRNA concentration to 200 ng / μL.

[0102] Next, a nanocarrier delivery system was prepared. Specifically, 20 mg / mL of poly(lactic-co-glycolic acid) (molecular weight 60 kDa), 3.5 mg / mL of polyethyleneimine (molecular weight 25 kDa), 5.5 mg / mL of polyethylene glycol (molecular weight 2 kDa), and 0.8 mg / mL of KALA targeting peptide were mixed, and a nanocarrier with an average particle size of 100 nm was prepared by the emulsion-solvent evaporation method. The surface potential of the obtained nanocarrier was +20 mV, which was beneficial for cell uptake. sgRNA and Cas9 protein were loaded into the nanocarrier, with the loading efficiency of sgRNA being 90% and that of Cas9 protein being 80%.

[0103] Then, precise transfection of petal cells was achieved through the nanocarrier delivery system. Specifically, in a transfection buffer containing 50 mM glucose, 130 mM NaCl, 4.5 mM KCl, 2.0 mM CaCl2, 1.0 mM MgCl2, and 20 mM HEPES (pH 7.3), 125 nM Cas9 protein, 175 nM sgRNA, and 30 μg / mL of the nanocarrier were added. A 30-μm diameter microneedle array was used to assist transfection, and transfection was carried out at 23.5 °C for 30 minutes. Preferably, the transfection efficiency achieved by this method reached 70% in the epidermal cells of pansy petals. This mechanical puncture-assisted transfection technique significantly improved the transfection efficiency and solved the problem that it was difficult to effectively transfect plant cells by traditional methods.

[0104] Subsequently, a dual-fluorescence reporter system was constructed to monitor the ANS gene expression level in real time. Specifically, a dual-fluorescence reporter vector pDF-ANS based on the pCAMBIA1300 backbone was designed. The vector contained 400-bp left and right border sequences each, the ANS gene promoter (-1500 to +50 bp) driving the expression of mCherry, the 35S promoter driving the expression of the reference fluorescent protein EGFP, the NOS terminator (289 bp), and the hygromycin resistance gene. A confocal microscopy imaging platform was constructed, equipped with 488-nm and 561-nm lasers, using a 60× / NA 1.4 oil immersion objective lens, a scanning speed of 400 Hz, a Z-axis step size of 0.5 μm, and an image resolution of 1024×1024 pixels. The ANS expression level was quantified by the mCherry / EGFP fluorescence intensity ratio method, monitored once every 12 hours for 8 consecutive days, and the monitoring data were the average values of 5 randomly selected 200-μm×200-μm regions. This dual-fluorescence reporter system can provide real-time and quantitative monitoring of gene expression levels, effectively excluding the interference of factors such as cell density and observation field differences.

[0105] Next, a temperature-inducible promoter was applied to control the activity of the FT gene. Specifically, a three-level temperature regulation system was designed based on the Arabidopsis HSP17.6 promoter, including a low-temperature promoter HSP-L with an induction temperature of 16.5 °C, a medium-temperature promoter HSP-M with an induction temperature of 23.5 °C, and a high-temperature promoter HSP-H with an induction temperature of 30 °C. The core region of the promoter consists of a heat shock response element 5'-nGAAnnTTCnnGAAn-3', a TATA box, and a transcription start site, and the total length of the regulatory sequence is 900 bp. HSP-L, HSP-M, and HSP-H were respectively coupled with the FT1, FT2, and FT3 genes. A temperature gradient induction system was used to control the flowering period, including: maintaining 16.5 °C for 0 - 14 days to activate HSP-L::FT1, maintaining 23.5 °C for 15 - 28 days to activate HSP-M::FT2, and maintaining 30 °C for 29 - 42 days to activate HSP-H::FT3. Each promoter-FT cascade system was equipped with different ANS editing sgRNA targeting sites. A PID controller was used to achieve a temperature regulation accuracy of ±0.4 °C. This temperature-inducible promoter system achieved precise regulation of flower blooming time, avoiding the complex operations of multiple transgenes required by traditional methods.

[0106] Finally, a rapid phenotypic detection device was integrated, and a microspectrophotometer was used to quantitatively measure the petal chromaticity value, and the CRISPR editing efficiency was optimized according to the measurement results. Specifically, a portable petal phenotype analysis system was constructed based on machine vision. The system includes a 24-megapixel high-resolution CMOS camera, an LED ring light with a color temperature of 5500 K, an autofocus macro lens with a working distance of 10 cm, an X-Rite ColorChecker standard color card, and a RaspberryPi 4B portable computing unit. Data processing was performed using automatic white balance and color calibration, petal contour extraction and segmentation, RGB, L*a*b*, HSV three-color system conversion and analysis, and an anthocyanin content prediction model. Data was collected every 18 hours, 5 angles were collected for each flower, and 3 technical replicates were collected for each angle. The parameters of L*, a*, b*, hue angle, saturation, and brightness values were measured. A nonlinear regression model was established to analyze the relationship between anthocyanin content and chromaticity value, and R 2 reached 0.95.

[0107] In this example, the microspectrophotometer system modified based on a Leica DM6B microscope includes a spectrophotometer module with a wavelength range of 400 - 700 nm and a resolution of 2 nm, a micro-region sampling device with an optional area of 60 μm 2 a CCD detector cooled at -80 °C, and a 16-bit ADC signal processing module. A mathematical model of petal chromaticity value and editing efficiency was established, including the calculation formula for the chromaticity change rate (ΔE) ΔE = [(ΔL*) 2+(Δa*) 2 +(Δb*) 2 (1 / 2) And the relationship formula between the editing efficiency (η) and ΔE is η = 1.38·ΔE / (12.45 + ΔE).

[0108] In this example, pansy seedlings at 7 weeks old were further selected and planted in flowerpots with a diameter of 15 cm. The growth substrate was peat:perlite:vermiculite = 5:3:2 (v / v / v), with a pH of 6.0, and the 1 / 2-strength Hoagland solution was irrigated twice a week. Three sgRNAs were designed for the ANS gene: sgRNA-ANS-E1 targeting the first exon caused complete inactivation of the ANS gene, sgRNA-ANS-E3 targeting the third exon caused partial inactivation of the ANS gene, and sgRNA-ANS-P targeting the promoter region down-regulated the expression of the ANS gene. sgRNA-ANS-E1 was coupled with HSP-L::FT1, sgRNA-ANS-E3 was coupled with HSP-M::FT2, and sgRNA-ANS-P was coupled with HSP-H::FT3 respectively, and the regulation of flower color transformation and staggered flowering periods was achieved through temperature induction. sgRNA-ANS-E1 caused the flower color to change from purple to white, sgRNA-ANS-E3 caused the flower color to change from purple to pink, and sgRNA-ANS-P caused the flower color to change from dark purple to light purple. The experimental results showed that 78% of the plants achieved a complete 42-day three-color staggered flowering cycle.

[0109] Preferably, the system integration uses microfluidic chip technology to integrate the delivery, monitoring, and feedback systems. The microfluidic chip is made by bonding PDMS and glass, and includes a cell culture area, a reagent input area, a monitoring area, and a waste liquid area. The channel width is 150 μm and the depth is 65 μm, which is used to realize the full automation operation from sgRNA delivery to phenotype monitoring. A multi-parameter response surface model was established to optimize the key parameters, including: adjusting the GC content of the sgRNA design to 50%, adjusting the surface charge of the nanocarrier to +20 mV, precisely controlling the temperature regulation within the range of the target temperature ±0.3 °C, and optimizing the sampling frequency from 24 hours to 12 hours. The system stability was manifested as the failure rate <4% during continuous operation for 42 days, and the data reproducibility was manifested as the coefficient of variation between phenotypic data groups <7%.

[0110] The experimental results showed that the CRISPR-mediated flower temporal expression regulation method based on the medium-concentration component ratio in this example had a 5 percentage point increase in transfection efficiency and a 5 percentage point increase in editing success rate compared with Example 1, with higher system stability, further improving the accuracy and reliability of flower flowering time sequence and flower color regulation.

[0111] Example 3: Flower Temporal Expression Regulation Method Based on High-Concentration Component Ratio ​

[0112] In this embodiment, a method for regulating the temporal expression of flowers mediated by CRISPR is provided. The method includes the following steps:

[0113] First, construct a database for editing key genes in flower development, and design an sgRNA library for the anthocyanin synthesis pathway. Based on PostgreSQL, a relational database is established, which collects genomic data, functional annotations, and expression profile systems of 2,546 key developmental genes from 12 major ornamental flower species. This database integrates a genomic data module, a regulatory network module, a phenotype analysis module, and an editing tool module. Through RNA-seq analysis, the spatio-temporal expression profiles of key enzyme-encoding genes in the anthocyanin synthesis pathway, such as CHS, CHI, F3H, F3'H, DFR, ANS, and 3GT, in the four stages S1-S4 of petal development are determined. The ATAC-seq technology is used to identify the open chromatin regions in the promoter regions of each gene, which are located in the interval from -2000 bp to +200 bp upstream of the transcription start site. The HMM model is used to predict the transcription factor binding sites, and the binding patterns of members of the MYB, bHLH, and WD40 transcription factor families are determined. On this basis, sgRNAs are designed for each key gene in the anthocyanin synthesis pathway, with a GC content of 60%, a spontaneous folding ΔG value of -18.0 kcal / mol, ≤1 off-target site, an sgRNA length of 20 nt, and a PAM sequence of the NGG type.

[0114] Subsequently, parallel synthesis of sgRNA templates is achieved by using chip oligonucleotide synthesis technology combined with PCR amplification. The T7 in vitro transcription system is used to generate sgRNA. The reaction system contains 2 μL of T7 RNA polymerase (50 U / μL), 20 μL of 5× transcription buffer, 8 μL of rNTP mixture (25 mM each), 5 μL of template DNA (100 ng / μL), 1 μL of RNase inhibitor (40 U / μL), 2 μL of DNase I (2 U / μL), and nuclease-free water is added to make up to 100 μL. After incubation at 37 °C for 4 hours, purification is carried out using the RNeasy MiniKit, and the final sgRNA concentration is adjusted to 200 ng / μL.

[0115] Next, a nanocarrier delivery system was prepared. Specifically, poly(lactic-co-glycolic acid) (molecular weight 75 kDa) at 25 mg / mL, polyethyleneimine (molecular weight 25 kDa) at 5 mg / mL, polyethylene glycol (molecular weight 2 kDa) at 8 mg / mL, and 1.2 mg / mL of KALA targeting peptide were mixed, and a nanocarrier with an average particle size of 120 nm was prepared by the emulsion-solvent evaporation method. The surface potential of the obtained nanocarrier was +25 mV, which was beneficial for cell uptake. sgRNA and Cas9 protein were loaded into the nanocarrier, with the loading efficiency of sgRNA being 95% and that of Cas9 protein being 85%.

[0116] Then, precise transfection of petal cells was achieved through the nanocarrier delivery system. Specifically, in a transfection buffer containing 55 mM glucose, 140 mM NaCl, 5.5 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, and 25 mM HEPES (pH 7.4), 150 nM Cas9 protein, 200 nM sgRNA, and 35 μg / mL of the nanocarrier were added. A 35-μm diameter microneedle array was used to assist transfection, and transfection was carried out at 25 °C for 35 minutes. Preferably, the transfection efficiency achieved by this method reached 75% in the epidermal cells of pansy petals. This mechanical puncture-assisted transfection technique significantly improved the transfection efficiency and solved the problem that it was difficult to effectively transfect plant cells by traditional methods.

[0117] Subsequently, a dual-fluorescent reporter system was constructed to monitor the ANS gene expression level in real time. Specifically, a dual-fluorescent reporter vector pDF-ANS based on the pCAMBIA1300 backbone was designed. The vector contained 400-bp left and right border sequences each, the ANS gene promoter (-1500 to +50 bp) driving the expression of mCherry, the 35S promoter driving the expression of the reference fluorescent protein EGFP, the NOS terminator (289 bp), and the hygromycin resistance gene. A confocal microscopy imaging platform was constructed, equipped with 488-nm and 561-nm lasers, using a 60× / NA 1.4 oil immersion objective, a scanning speed of 400 Hz, a Z-axis step size of 0.5 μm, and an image resolution of 1024×1024 pixels. The ANS expression level was quantified by the mCherry / EGFP fluorescence intensity ratio method, monitored once every 12 hours for 10 consecutive days, and the monitoring data were the average values of 5 randomly selected 200-μm×200-μm regions. This dual-fluorescent reporter system can provide real-time and quantitative monitoring of gene expression levels, effectively excluding the interference of factors such as cell density and observation field differences.

[0118] Next, a temperature-inducible promoter was applied to control the activity of the FT gene. Specifically, a three-level temperature regulation system was designed based on the Arabidopsis HSP17.6 promoter, including a low-temperature promoter HSP-L with an induction temperature of 18°C, a medium-temperature promoter HSP-M with an induction temperature of 25°C, and a high-temperature promoter HSP-H with an induction temperature of 32°C. The core region of the promoter consists of a heat shock response element 5'-nGAAnnTTCnnGAAn-3', a TATA box, and a transcription start site, and the total length of the regulatory sequence is 1000 bp. HSP-L, HSP-M, and HSP-H were respectively coupled with the FT1, FT2, and FT3 genes. A temperature gradient induction system was used to control the flowering period, including: maintaining 18°C for 0-14 days to activate HSP-L::FT1, maintaining 25°C for 15-28 days to activate HSP-M::FT2, and maintaining 32°C for 29-42 days to activate HSP-H::FT3. Each promoter-FT cascade system was equipped with different ANS editing sgRNA targeting sites. A PID controller was used to achieve a temperature regulation accuracy of ±0.3°C. This temperature-inducible promoter system achieved precise regulation of the flowering time of flowers, avoiding the complex operations of multiple transgenes required by traditional methods.

[0119] Finally, a rapid phenotypic detection device was integrated, and a microspectrophotometer was used to quantitatively measure the chromaticity value of the petals, and the CRISPR editing efficiency was optimized according to the measurement results. Specifically, a portable petal phenotypic analysis system was constructed based on machine vision. The system includes a 24-megapixel high-resolution CMOS camera, an LED ring light with a color temperature of 5500K, an autofocus macro lens with a working distance of 15 cm, an X-Rite ColorChecker standard color card, and a RaspberryPi 4B portable computing unit. Data processing was performed using automatic white balance and color calibration, petal contour extraction and segmentation, RGB, L*a*b*, HSV three-color system conversion and analysis, and an anthocyanin content prediction model. Data was collected once every 12 hours, 5 angles were collected for each flower, and 3 technical replicates were collected for each angle. The parameters of L*, a*, b*, hue angle, saturation, and brightness values were measured. A nonlinear regression model was established to analyze the relationship between anthocyanin content and chromaticity value, and R 2 reached 0.98.

[0120] In this example, the microspectrophotometer system modified based on the Leica DM6B microscope includes a spectrophotometer module with a wavelength range of 400-700 nm and a resolution of 2 nm, a micro-region sampling device with an optional area of 100 μm 2 , a CCD detector cooled at -80°C, and a 16-bit ADC signal processing module. A mathematical model of petal chromaticity value and editing efficiency was established, including the calculation formula for the chromaticity change rate (ΔE) ΔE = [(ΔL*) 2 +(Δa*)2 +(Δb*) 2 (1 / 2) And the formula for the relationship between the editing efficiency (η) and ΔE is η = 1.50·ΔE / (11.10 + ΔE).

[0121] In this example, pansy seedlings at 8 weeks old were further selected and planted in flowerpots with a diameter of 15 cm. The growth substrate was peat:perlite:vermiculite = 5:3:2 (v / v / v), with a pH of 6.2, and the 1 / 2 strength Hoagland solution was irrigated twice a week. Three sgRNAs were designed for the ANS gene: sgRNA-ANS-E1 targeting the first exon caused complete inactivation of the ANS gene, sgRNA-ANS-E3 targeting the third exon caused partial inactivation of the ANS gene, and sgRNA-ANS-P targeting the promoter region down-regulated the expression of the ANS gene. sgRNA-ANS-E1 was coupled with HSP-L::FT1, sgRNA-ANS-E3 was coupled with HSP-M::FT2, and sgRNA-ANS-P was coupled with HSP-H::FT3 respectively, and the regulation of flower color transformation and staggered flowering periods was achieved through temperature induction. sgRNA-ANS-E1 caused the flower color to change from purple to white, sgRNA-ANS-E3 caused the flower color to change from purple to pink, and sgRNA-ANS-P caused the flower color to change from dark purple to light purple. The experimental results showed that 85% of the plants achieved a complete 42-day three-color staggered flowering cycle.

[0122] Preferably, the system integration adopts microfluidic chip technology to integrate the delivery, monitoring, and feedback systems. The microfluidic chip is made by bonding PDMS and glass, and includes a cell culture area, a reagent input area, a monitoring area, and a waste liquid area. The channel width is 200 μm and the depth is 80 μm, which is used to realize the full-automatic operation from sgRNA delivery to phenotype monitoring. A multi-parameter response surface model was established to optimize the key parameters, including: adjusting the GC content of sgRNA design to 55%, adjusting the surface charge of the nanocarrier to +22 mV, precisely controlling the temperature regulation within the range of the target temperature ±0.3 °C, and optimizing the sampling frequency from 24 hours to 12 hours. The system stability was manifested as the failure rate < 3% during continuous operation for 42 days, and the data reproducibility was manifested as the coefficient of variation between phenotype data groups < 6%.

[0123] The experimental results showed that the CRISPR-mediated flower temporal expression regulation method based on the high-concentration component ratio in this example had higher transfection efficiency, higher editing success rate, and better system stability compared with Example 1 and Example 2, further improving the accuracy and reliability of flower flowering time sequence and flower color regulation. However, it should be noted that although the high-concentration component ratio improved the editing efficiency and system stability, it also increased the material cost and operation complexity, and a trade-off needs to be made according to specific requirements in practical applications. ​

[0124] Example 4: Floral Temporal Expression Regulation Method Based on Orthogonal Combination Optimization

[0125] In this example, the orthogonal experimental design method is used to optimize the key parameter combination of the CRISPR-mediated floral temporal expression regulation method. This method includes the following steps:

[0126] First, construct a key gene editing database for flower development, and design an sgRNA library for the anthocyanin synthesis pathway. Based on PostgreSQL, a relational database is established, which includes genomic data, functional annotations, and expression profiles of 2,546 key development genes from 12 major ornamental flower species. This database integrates genomic data modules, regulatory network modules, phenotype analysis modules, and editing tool modules. Through RNA-seq analysis, the spatio-temporal expression profiles of key enzyme-encoding genes in the anthocyanin synthesis pathway, such as CHS, CHI, F3H, F3'H, DFR, ANS, and 3GT, at four stages of petal development S1-S4 are determined. The ATAC-seq technology is used to identify the open chromatin regions in the promoter regions of each gene, which are located in the interval from -2000bp to +200bp upstream of the transcription start site. The HMM model is used to predict the transcription factor binding sites, and the binding patterns of members of the MYB, bHLH, and WD40 transcription factor families are determined. On this basis, sgRNAs are designed for each key gene in the anthocyanin synthesis pathway, with a GC content of 55%, a spontaneous folding ΔG value of -15.0 kcal / mol, ≤1 off-target site, an sgRNA length of 20nt, and a PAM sequence of the NGG type.

[0127] Subsequently, the parallel synthesis of sgRNA templates is achieved by using chip oligonucleotide synthesis technology combined with PCR amplification. The T7 in vitro transcription system is used to generate sgRNA, and the reaction system contains 2μL T7 RNA polymerase (50U / μL), 20μL 5× transcription buffer, 8μL rNTP mixture (25mM each), 5μL template DNA (100ng / μL), 1μL RNase inhibitor (40U / μL), 2μL DNase I (2U / μL), and nuclease-free water is added to make up to 100μL. After incubation at 37°C for 4 hours, it is purified using the RNeasy MiniKit, and the final sgRNA concentration is adjusted to 200ng / μL.

[0128] Next, the component ratios of the nanocarrier delivery system were optimized through orthogonal experimental design. Through the L9(3^4) orthogonal experimental design and analysis, the optimal component ratios were determined as follows: 22 mg / mL of poly(lactic-co-glycolic acid) (molecular weight 65 kDa), 4 mg / mL of polyethyleneimine (molecular weight 25 kDa), 6 mg / mL of polyethylene glycol (molecular weight 2 kDa), and 1.0 mg / mL of KALA targeting peptide. The nanocarriers with an average particle size of 110 nm were prepared by the emulsion-solvent evaporation method. The surface potential of the obtained nanocarriers was +20 mV, which was beneficial for cell uptake. SgRNA and Cas9 protein were loaded into the nanocarriers, with the loading efficiency of sgRNA being 92% and that of Cas9 protein being 82%.

[0129] Then, precise transfection of petal cells was achieved through the nanocarrier delivery system. The transfection conditions were optimized by the response surface method, and the optimal conditions were determined as follows: in the transfection buffer containing 52 mM glucose, 135 mM NaCl, 4.8 mM KCl, 2.2 mM CaCl2, 1.1 mM MgCl2, and 22 mM HEPES (pH 7.35), 135 nM Cas9 protein, 185 nM sgRNA, and 32 μg / mL of nanocarriers were added. A 32-μm diameter microneedle array was used to assist transfection, and transfection was performed at 24 °C for 32 minutes. Preferably, the transfection efficiency achieved by this method reached 72% in the epidermal cells of pansy petals.

[0130] Subsequently, a dual-fluorescence reporting system was constructed to monitor the ANS gene expression level in real time. Specifically, a dual-fluorescence reporting vector pDF-ANS based on the pCAMBIA1300 backbone was designed. The vector contained 400-bp left and right border sequences each, the ANS gene promoter (-1500 to +50 bp) driving the expression of mCherry, the 35S promoter driving the expression of the reference fluorescent protein EGFP, the NOS terminator (289 bp), and the hygromycin resistance gene. A confocal microscopy imaging platform was constructed, equipped with 488-nm and 561-nm lasers, using a 60× / NA 1.4 oil immersion objective, with a scanning speed of 400 Hz, a Z-axis step size of 0.5 μm, and an image resolution of 1024×1024 pixels. The ANS expression level was quantified by the mCherry / EGFP fluorescence intensity ratio method, monitored once every 12 hours for 9 consecutive days, and the monitoring data were the average values of 5 randomly selected 200-μm×200-μm regions.

[0131] Next, optimize the temperature-inducible promoter control system. Optimize the three-level temperature regulation system through central composite design, and determine the optimal induction temperatures as follows: the low-temperature promoter HSP-L is 17 °C, the medium-temperature promoter HSP-M is 24 °C, and the high-temperature promoter HSP-H is 31 °C. The core region of the promoter consists of a heat shock response element 5'-nGAAnnTTCnnGAAn-3', a TATA box, and a transcription start site, and the total length of the regulatory sequence is 950 bp. Couple HSP-L, HSP-M, and HSP-H with FT1, FT2, and FT3 genes respectively. Use a temperature gradient induction system to control the flowering period, including: maintaining 17 °C for 0 - 14 days to activate HSP-L::FT1, maintaining 24 °C for 15 - 28 days to activate HSP-M::FT2, and maintaining 31 °C for 29 - 42 days to activate HSP-H::FT3. Each promoter-FT cascade system is equipped with different ANS editing sgRNA targeting sites. Use a PID controller to achieve a temperature control accuracy of ±0.3 °C.

[0132] Finally, integrate a rapid phenotypic detection device, use a microspectrophotometer to quantitatively measure the petal chromaticity value, and optimize the CRISPR editing efficiency according to the measurement results. Specifically, construct a portable petal phenotype analysis system based on machine vision. The system includes a 24-megapixel high-resolution CMOS camera, an LED ring light with a color temperature of 5500 K, an autofocus macro lens with a working distance of 12 cm, an X-Rite ColorChecker standard color card, and a RaspberryPi 4B portable computing unit. Data processing is performed using automatic white balance and color calibration, petal contour extraction and segmentation, RGB, L*a*b*, HSV three-color system conversion and analysis, and an anthocyanin content prediction model. Data is collected once every 12 hours, 5 angles are collected for each flower, and 3 technical replicates are collected for each angle. Parameters such as L*, a*, b*, hue angle, saturation, and brightness values are measured. Establish a non-linear regression model to analyze the relationship between anthocyanin content and chromaticity value, with R 2 reaching 0.97.

[0133] In this embodiment, the microspectrophotometer system modified based on a Leica DM6B microscope includes a spectrophotometer module with a wavelength range of 400 - 700 nm and a resolution of 2 nm, a micro-region sampling device with an optional area of 75 μm 2 , a CCD detector cooled at -80 °C, and a 16-bit ADC signal processing module. Establish a mathematical model of petal chromaticity value and editing efficiency, including the calculation formula for the chromaticity change rate (ΔE) ΔE = [(ΔL*) 2 +(Δa*) 2 +(Δb*) 2 (1 / 2) ​The formula for the relationship between editing efficiency (η) and ΔE is η = 1.45·ΔE / (12.00 + ΔE).

[0134] In this example, pansy seedlings at the age of 7 - 8 weeks were further selected and planted in flowerpots with a diameter of 15 cm. The growth substrate was peat:perlite:vermiculite = 5:3:2 (v / v / v), with a pH of 6.1, and the 1 / 2 strength Hoagland solution was irrigated twice a week. Three sgRNAs were designed for the ANS gene: sgRNA - ANS - E1 targeting the first exon caused complete inactivation of the ANS gene, sgRNA - ANS - E3 targeting the third exon caused partial inactivation of the ANS gene, and sgRNA - ANS - P targeting the promoter region down - regulated the expression of the ANS gene. sgRNA - ANS - E1 was coupled with HSP - L::FT1, sgRNA - ANS - E3 was coupled with HSP - M::FT2, and sgRNA - ANS - P was coupled with HSP - H::FT3 respectively, and the regulation of flower color transformation and staggered flowering periods was achieved through temperature induction. The experimental results showed that 82% of the plants achieved a complete 42 - day three - color staggered flowering cycle.

[0135] Preferably, the system integration uses microfluidic chip technology to integrate the delivery, monitoring, and feedback systems. The microfluidic chip is made by bonding PDMS and glass, and includes a cell culture area, a reagent input area, a monitoring area, and a waste liquid area. The channel width is 175 μm and the depth is 65 μm, which is used to realize the full - automation operation from sgRNA delivery to phenotype monitoring. A multi - parameter response surface model was established to optimize the key parameters, including: adjusting the GC content of sgRNA design to 52%, adjusting the surface charge of the nanocarrier to +20 ± 1 mV, precisely controlling the temperature regulation within the range of the target temperature ±0.3 °C, and optimizing the sampling frequency to 12 hours. The system stability was shown as the failure rate < 3.5% during continuous operation for 42 days, and the data reproducibility was shown as the coefficient of variation of phenotypic data between groups < 6.5%.

[0136] The experimental results showed that the CRISPR - mediated floral temporal expression regulation method based on orthogonal combination optimization in this example achieved, through systematic optimization of each key parameter, an effect close to that of the high - concentration component ratio method while maintaining a relatively low material cost. The transfection efficiency reached 72%, the editing success rate reached 82%, the system had good stability and high data reproducibility, providing a more economical and efficient technical solution for the regulation of floral flowering time and flower color.

Claims

1. A CRISPR-mediated method for regulating temporal expression of flowers, characterized in that: The method comprises the following steps: a) Construct a database of key gene editing in flower development and design a sgRNA library targeting anthocyanin synthesis pathway; b) preparing a nanocarrier delivery system, comprising: i) mixing 15-25 mg / mL of poly(lactic acid-co-glycolic acid), 2-5 mg / mL of polyethyleneimine, 3-8 mg / mL of polyethylene glycol and 0.5-1.2 mg / mL of the targeting peptide, and preparing a nanocarrier with an average particle size of 80-120 nm by an emulsification-solvent evaporation method; ii) loading sgRNA and Cas9 protein in the nanocarrier, wherein the sgRNA loading efficiency is ≥85%, and the Cas9 protein loading efficiency is ≥75%; c) achieving precise transfection of petal cells by the nanocarrier delivery system, wherein the precise transfection comprises: i) adding 100-150 nM Cas9 protein, 150-200 nM sgRNA and 25-35 μg / mL nanocarrier to a transfection buffer containing 45-55 mM glucose, 120-140 mM NaCl, 3.5-5.5 mM KCl, 1.5-2.5 mM CaCl2, 0.8-1.2 mM MgCl2 and 15-25 mM HEPES (pH 7.2-7.4); ii) using a 25-35 μm diameter microneedle array to assist transfection at 22-25°C for 25-35 minutes; d) constructing a dual fluorescence reporter system to monitor the ANS gene expression level in real time; e) Using a temperature-inducible promoter to control FT gene activity, flowering period regulation was achieved through a temperature gradient induction system; f) An integrated phenotypic rapid detection device was used to quantitatively measure the color value of the petals using a microspectrophotometer, and the CRISPR editing efficiency was optimized based on the measurement results.

2. The method according to claim 1, characterized in that The step a) comprises: i) establishing a relational database based on PostgreSQL, including flower genome data, functional annotation and expression spectrum system, wherein the database integrates genome data module, regulatory network module, phenotypic analysis module and editing tool module; ii) Determine the spatiotemporal expression profiles of genes encoding key enzymes in the anthocyanin biosynthesis pathway, including CHS, CHI, F3H, F3'H, DFR, ANS, and 3GT, during the four stages of petal development, S1-S4, by RNA-seq analysis; iii) ATAC-seq was used to identify the open chromatin region in the promoter region of each gene, localized in the interval from -2000 bp to +200 bp upstream of the transcription start site; iv) Predict transcription factor binding sites using the HMM model and determine the binding patterns of MYB, bHLH and WD40 transcription factor family members; v) Design 8-15 sgRNAs for each key gene of anthocyanin biosynthesis pathway, with a GC content of 40-60%, a spontaneous folding ΔG ≥ -12.0 kcal / mol, ≤ 2 off-target sites, a sgRNA length of 20 nt, and a PAM sequence of NGG type; vi) Using chip oligonucleotide synthesis technology combined with PCR amplification to achieve parallel synthesis of sgRNA templates; vii) sgRNA was generated using a T7 in vitro transcription system containing 2 μL T7 RNA polymerase (50 U / μL), 20 μL 5× transcription buffer, 8 μL rNTP mixture (25 mM each), 5 μL template DNA (100 ng / μL), 1 μL RNase inhibitor (40 U / μL), 2 μL DNase I (2 U / μL) and nuclease-free water to 100 μL. After incubation at 37°C for 4 hours, sgRNA was purified using RNeasy MiniKit and the final sgRNA concentration was adjusted to 200 ng / μL.

3. The method according to claim 1, characterized in that: The step d) comprises: i) Designing a dual fluorescence reporter vector pDF-ANS based on the pCAMBIA1300 backbone, the vector comprises 400 bp of left and right border sequences, an ANS gene promoter (-1500 to +50 bp) driving mCherry expression, a 35S promoter driving reference fluorescent protein EGFP expression, a NOS terminator (289 bp) and a hygromycin resistance gene; ii) Construct a confocal microscopy imaging platform, configure 488 nm and 561 nm lasers, use a 60× / NA 1.4 oil objective, a scanning speed of 400 Hz, a Z-axis step size of 0.5 μm, and an image resolution of 1024×1024 pixels; iii) quantification of ANS expression levels using the mCherry / EGFP fluorescence intensity ratio method; iv) Monitor once every 12 hours for 7-10 days, and use the average value of 5 randomly selected 200μm×200μm areas as the monitoring data.

4. The method according to claim 1, characterized in that: The step e) comprises: i) designing a three-level temperature regulation system based on the Arabidopsis thaliana HSP17.6 promoter, including a low-temperature promoter HSP-L with an induction temperature of 15-18°C, a medium-temperature promoter HSP-M with an induction temperature of 22-25°C, and a high-temperature promoter HSP-H with an induction temperature of 28-32°C, wherein the core region of the promoter comprises a heat shock response element 5'-nGAAnnTTCnnGAAn-3', a TATA box, and a transcription initiation site, and the total length of the regulatory sequence is 800-1000 bp; ii) coupling HSP-L, HSP-M, and HSP-H to FT1, FT2, and FT3 genes, respectively; iii) using a temperature gradient induction system to control flowering time, including: maintaining 15-18°C to activate HSP-L::FT1 during 0-14 days, maintaining 22-25°C to activate HSP-M::FT2 during 15-28 days, and maintaining 28-32°C to activate HSP-H::FT3 during 29-42 days; iv) Each promoter-FT cascade system is equipped with a different ANS editing sgRNA targeting site; v) Use PID controller to achieve temperature control accuracy of ±0.5℃.

5. The method according to claim 1, characterized in that The step f) comprises: i) constructing a portable petal phenotyping system based on machine vision, the system comprising a 24-megapixel high-resolution CMOS camera, an LED ring light source with a color temperature of 5500K, an autofocus macro lens with a working distance of 5-15cm, an X-RiteColorChecker standard color card, and a RaspberryPi 4B portable computing unit; ii) Data processing using automatic white balance and color calibration, petal contour extraction and segmentation, RGB, L*a*b*, HSV color system conversion and analysis, and anthocyanin content prediction model; iii) Data were collected every 24 hours, 5 angles were collected for each flower, and 3 technical replicates were performed for each angle; iv) Determination of L* (lightness, 0-100), a* (red-green axis, -128 to +127), b* (yellow-blue axis, -128 to +127), hue angle (0-360°), saturation (0-100%) and lightness value (0-100%); v) Establish a nonlinear regression model to analyze the relationship between anthocyanin content and chromaticity value, R 2 ≥0.

92.

6. The method according to claim 1, characterized in that The method comprises the steps of staggered regulation of the flowering period of individual pansy plants: i) 6-8 week old pansy seedlings were selected and planted in 15 cm diameter pots, with a growth medium of peat:perlite:vermiculite=5:3:2 (v / v / v), pH 5.8-6.2, and watered twice a week with 1 / 2 strength Hoagland solution; ii) Three sgRNAs were designed for the ANS gene: sgRNA-ANS-E1 targeting exon 1 resulted in complete inactivation of the ANS gene, sgRNA-ANS-E3 targeting exon 3 resulted in partial inactivation of the ANS gene, and sgRNA-ANS-P targeting the promoter region downregulated ANS gene expression; iii) sgRNA-ANS-E1 was coupled with HSP-L::FT1, sgRNA-ANS-E3 with HSP-M::FT2, and sgRNA-ANS-P with HSP-H::FT3, respectively, to achieve flower color transition and staggered flowering period regulation through temperature induction.

7. A CRISPR-mediated floral temporal expression control system for implementing the method of claim 1, characterized in that: The system comprises: a) Database of key gene editing in floral development and sgRNA library; b) a nanocarrier delivery system, comprising a nanocarrier prepared from poly(lactic-co-glycolic acid), polyethyleneimine, polyethylene glycol and a targeting peptide; c) Dual fluorescence reporter system, including dual fluorescence reporter vector pDF-ANS and confocal microscopy imaging platform; d) Temperature-inducible promoter control system, including a three-level temperature control system and a temperature gradient induction system; e) phenotypic rapid detection devices, including portable petal phenotyping systems based on machine vision; f) a microspectrophotometer quantitative feedback system, including a microspectrophotometer and a CRISPR editing efficiency optimization module; g) Microfluidic chips for integrating delivery, monitoring and feedback systems.

8. The system according to claim 7, characterized in that The nanocarrier delivery system comprises: i) a nanocarrier prepared by an emulsification-solvent evaporation method from 15-25 mg / mL of poly(lactic-co-glycolic acid) (molecular weight 45-75 kDa), 2-5 mg / mL of polyethyleneimine (molecular weight 25 kDa), 3-8 mg / mL of polyethylene glycol (molecular weight 2 kDa), and 0.5-1.2 mg / mL of KALA targeting peptide, with an average particle size of 80-120 nm and a surface potential of +15 to +25 mV; ii) Mechanical puncture-assisted transfection device, including a microneedle array with a diameter of 25-35 μm; iii) Transfection system, including transfection buffer, 100-150 nM Cas9 protein, 150-200 nM sgRNA and 25-35 μg / mL nanocarrier.

9. The system according to claim 7, characterized in that The microspectrophotometer quantitative feedback system comprises: i) A microspectrophotometer system based on a Leica DM6B microscope, including a spectrophotometer module with a wavelength range of 400-700nm and a resolution of 2nm, and an optional area of ​​25-100μm 2 Micro-area sampling device, -80℃ cryogenically cooled CCD detector and 16-bit ADC signal processing module; ii) A mathematical model of petal chromaticity value and editing efficiency, including the chromaticity change rate (ΔE) calculation formula ΔE = [(ΔL*) 2 +(Δa*) 2 +(Δb*) 2 ] (1 / 2) The relationship between editing efficiency (η) and ΔE is η = k1·ΔE / (k2+ΔE), where k1 = 1.38 ± 0.12, k2 = 12.45 ± 1.35; iii) CRISPR editing efficiency optimization module for adjusting sgRNA target position based on chromatic feedback, dynamically adjusting Cas9 concentration (80-200 nM) and adjusting nanoparticle surface charge (+15 to +30 mV).

10. The system according to claim 7, characterized in that The microfluidic chip is made of PDMS bonded to glass, and includes a cell culture area, a reagent input area, a monitoring area and a waste liquid area. The channel width is 100-200μm and the depth is 50-80μm. It is used to achieve fully automated operations from sgRNA delivery to phenotypic monitoring, and optimize key parameters through a multi-parameter response surface model, including: adjusting the GC content of the sgRNA design to 45-55%, adjusting the surface charge of the nanocarrier to +20±2mV, precisely controlling the temperature within the target temperature range of ±0.3°C, and optimizing the sampling frequency from 24 hours to 12 hours.