Carboxylated fluorescent quantum dot-loaded gene editing system and its application in gene editing of aquatic animals

Abstract

This invention relates to the field of aquaculture technology, specifically to a carboxyl fluorescent quantum dot-loaded gene editing system and its application in gene editing of aquatic animals. The invention provides a method for preparing the carboxyl fluorescent quantum dot-loaded gene editing system, including the preparation of functionalized carboxyl fluorescent quantum dots and the ligation of functionalized carboxyl fluorescent quantum dots to plasmid DNA, resulting in a carboxyl fluorescent quantum dot-loaded gene editing system, which can then be used for gene editing of fertilized eggs or cell transfection in aquatic animals. This invention utilizes safe and efficient eggshell digestive enzymes and nanomaterials for gene editing breeding in aquatic animals.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, specifically to a carboxyl fluorescent quantum dot-loaded gene editing system and its application in gene editing of aquatic animals. Background Technology

[0002] In recent years, the aquaculture industry has flourished, but the breeding of aquatic animals faces numerous challenges. Grass carp (Ctenopharyngodon idellus), largemouth bass (Micropterus salmoides), and striped grouper (Epinephelus coioides) are important economic aquatic animals. Meanwhile, zebrafish (Danio rerio) and rare gudgeon (Gobiocypris rarus) are important model animals, widely used in various research experiments. Gene editing breeding technology for aquatic animals is of great significance to the development of the aquaculture industry, serving as an important means to improve the quality of aquatic seed products. Developing novel gene editing methods for important economic animals and model animals is of great importance. Therefore, developing efficient, large-scale gene editing delivery platforms for aquatic animals is extremely important for improving the development of the aquatic seed industry.

[0003] Traditional microinjection for gene editing is inefficient, limited by equipment, and prone to causing embryo rupture, severely impacting hatching rates in aquatic animal embryos. Therefore, developing novel, large-scale, and efficient gene editing systems is crucial. The eggshell of an aquatic animal fertilized egg is a vital structure for maintaining embryonic environmental stability and plays a significant role in embryonic development. While nanomaterials are widely used in drug delivery, their application in aquatic animal gene editing delivery has not yet been realized. The presence of the eggshell hinders the entry of nanomaterials or biomolecules into the embryo; therefore, disrupting the eggshell of aquatic animal embryos is essential for nanomaterial plasmid delivery systems.

[0004] Therefore, finding safe and efficient eggshell digestive enzymes and nanomaterials is urgently needed for gene editing breeding of aquatic animals. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a carboxyl fluorescent quantum dot loaded gene editing system and its preparation method. This invention uses safe and efficient eggshell digestive enzymes and nanomaterials for gene editing breeding of aquatic animals.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a method for preparing a carboxyl fluorescent quantum dot-loaded gene editing system, the method comprising preparing functionalized carboxyl fluorescent quantum dots and ligating the functionalized carboxyl fluorescent quantum dots to plasmid DNA; wherein...

[0008] The preparation of the functionalized carboxyl fluorescent quantum dots includes:

[0009] S1. Disperse carboxyl fluorescent quantum dots in pure water, add N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and stir the reaction at room temperature to activate the carboxyl groups on the surface of the carboxyl fluorescent quantum dots;

[0010] S2. Branched polyethyleneimine was added to the carboxyl-activated carboxyl fluorescent quantum dot reaction system, and the reaction was stirred at room temperature;

[0011] S3. The reaction system was dialyzed using a 7000D dialysis bag to remove unreacted N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and branched polyethyleneimine. The resulting branched polyethyleneimine functionalized carboxyl fluorescent quantum dots were functionalized carboxyl fluorescent quantum dots and stored at 4℃.

[0012] The connection between the functionalized carboxyl fluorescent quantum dots and plasmid DNA includes:

[0013] Plasmid DNA was diluted in deionized water, and functionalized carboxyl fluorescent quantum dots that had been sonicated were added to the solution. The mixture was then incubated at room temperature with shaking to obtain a carboxyl fluorescent quantum dot-loaded gene editing system, which was subsequently used for gene editing of fertilized eggs or cell transfection in aquatic animals.

[0014] Preferred,

[0015] The preparation of the functionalized carboxyl fluorescent quantum dots includes:

[0016] S1. Carboxyl fluorescent quantum dots with a carboxyl content of 6 μM / mL were dispersed in pure water at 0.05 μM, and N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added to a final concentration of 12 μM / mL. The mixture was stirred at room temperature for 2 h to activate the carboxyl groups on the surface of the carboxyl fluorescent quantum dots.

[0017] S2. Add 60 μM / mL branched polyethyleneimine to the carboxyl-activated carboxyl fluorescent quantum dot reaction system and stir at room temperature for 24 h;

[0018] S3. The reaction system was dialyzed using a 7000D dialysis bag to remove unreacted N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and branched polyethyleneimine. The resulting branched polyethyleneimine functionalized carboxyl fluorescent quantum dots are functionalized carboxyl fluorescent quantum dots. They do not aggregate within 90 days when stored at 4°C.

[0019] The connection between the functionalized carboxyl fluorescent quantum dots and plasmid DNA includes:

[0020] Plasmid DNA with a final concentration of 100 ng / μL was diluted in 100 μL of deionized water. 100-400 μL of functionalized carboxyl fluorescent quantum dots that had been sonicated (500 W, 3-5 s) were added to the solution. The mixture was then incubated at 150 rpm for 30 min at room temperature to obtain a carboxyl fluorescent quantum dot-loaded gene editing system, which was subsequently used for gene editing of fertilized eggs or cell transfection in aquatic animals.

[0021] Preferably, the ratio of the number of nitrogen atoms N in the branched polyethyleneimine to the number of negative charges P in the plasmid DNA is in the range of 10-14:1.

[0022] Preferably, the particle size of the carboxyl fluorescent quantum dots is 1-10 nm; the molecular weight of the branched polyethyleneimine is 900 g / M; and the pH of the branched polyethyleneimine is 7.

[0023] Secondly, a carboxyl fluorescent quantum dot-loaded gene editing system is provided, which is prepared by the preparation method described in this invention.

[0024] Thirdly, the present invention provides the application of the carboxyl fluorescent quantum dot loaded gene editing system in gene editing of fertilized eggs of aquatic animals, wherein the eggshell of the fertilized egg is digested by an enzyme preparation.

[0025] Preferably, the aquatic animals include zebrafish, rare gudgeon, grass carp, largemouth bass, and oblique grouper; the enzyme preparation is selected from one or more of trypsin, streptomycin, papain, keratinase, and chymotrypsin.

[0026] Preferably, the carboxyl fluorescent quantum dot loaded gene editing system is delivered to aquatic animal fertilized eggs by immersion in a bath, and the shell-removed aquatic animal fertilized eggs are immersed in the carboxyl fluorescent quantum dot loaded gene editing system for 0.5-2 hours.

[0027] Preferably, the application concentration of the carboxyl fluorescent quantum dot-loaded gene editing system is 1-50 mg / L.

[0028] Fourthly, the present invention provides the application of the carboxyl fluorescent quantum dot-loaded gene editing system in aquatic animal cell lines.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] Fluorescent quantum dots are a novel type of nanoparticle, a semiconductor nanoparticle composed of group 0 elements capable of fluorescing upon excitation light. They possess unique quantum size and surface effects, are easily modifiable, exhibit excellent optical properties, and demonstrate good biosafety and biocompatibility. The fluorescent quantum dots used in this invention have extremely high potential and application value in delivering nucleic acids for gene editing in aquatic animals, providing a new strategy and method for gene editing in aquatic animals.

[0031] The method for digesting the eggshells of fertilized aquatic animal eggs using enzyme preparations provided by this invention includes the combined use of papain, keratinase, chymotrypsin, trypsin, and streptomycin, which removes the first obstacle for delivering gene editing systems using nanomaterials.

[0032] This invention provides the activation of N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to obtain carboxyl-activated carboxyl fluorescent quantum dots, making them nanoparticles capable of supporting gene editing systems. Furthermore, this invention provides PEI (polyethyleneimine) functionalization modification of fluorescent quantum dots to further enhance their ability to support gene editing systems, constructing nanocomposites for gene editing of aquatic animal zygotes. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of carboxyl fluorescent quantum dots.

[0034] Figure 2 To observe the expression of exogenous GFP gene delivered in EPC cells by a carboxyl fluorescent quantum dot-loaded gene editing system (plasmid loaded with GFP gene) using fluorescence microscopy.

[0035] Figure 3 To deliver exogenous GFP gene expression in zebrafish eggs using a carboxylated fluorescent quantum dot-loaded gene editing system (plasmid loaded with GFP gene).

[0036] Figure 4 Delivery of the tyr gene to a carboxylated fluorescent quantum dot-loaded gene editing system (plasmid loaded with the tyr gene) caused a change in zebrafish body color.

[0037] Figure 5 The tyr gene sequence mutation in zebrafish was induced by a carboxyl fluorescent quantum dot-loaded gene editing system (plasmid loaded with the tyr gene).

[0038] in, Figures 2-5 The control group in this context refers to fluorescent quantum dots without carboxyl groups. Detailed Implementation

[0039] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention clearer. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0040] In this embodiment of the invention, the carboxyl fluorescent quantum dots were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., with product number F122252.

[0041] In this embodiment of the invention, a method for functionalizing carboxyl fluorescent quantum dots is provided, the specific steps of which are as follows:

[0042] S1. Carboxyl fluorescent quantum dots (carboxyl content of 6 μM / mL) were dispersed in pure water at 0.05 μM, and NHS (N-hydroxysuccinimide) and EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) were added to a final concentration of 12 μM / mL. The mixture was stirred at room temperature for 2 h to activate the carboxyl groups on the surface of the carboxyl fluorescent quantum dots.

[0043] S2. Add 60 μM / mL of branched polyethyleneimine (molecular weight 900 g / M, pH=7) to the carboxyl-activated carboxyl fluorescent quantum dot reaction system and stir at room temperature for 24 h.

[0044] The carboxyl fluorescent quantum dots have a particle size of 1-10 nm; the branched polyethyleneimine has a molecular weight of 900 g / M; and the branched polyethyleneimine has a pH of 7.

[0045] S3. The reaction system was dialyzed using a 7000D dialysis bag to remove unreacted NHS, EDC, and branched polyethyleneimine. The PEI-functionalized quantum dots stored at 4°C did not aggregate within 90 days. Figure 1 As shown.

[0046] In this embodiment of the invention, a method for loading and linking PEI-functionalized carboxyl fluorescent quantum dots to plasmid DNA is provided, wherein PEI-functionalized quantum dots and plasmid DNA are linked by non-covalent bonds, and the method is as follows:

[0047] S4. Dilute the plasmid DNA to a final concentration of 100 ng / μL in 100 μL of deionized water, add 100-400 μL of PEI-functionalized carboxyl fluorescent quantum dots after sonication, and incubate at room temperature with shaking at 150 rpm for 30 min. It can then be used for fish egg gene editing or cell transfection.

[0048] The ratio of the number of nitrogen atoms N in the branched polyethyleneimine to the number of negative charges P in the plasmid DNA is in the range of 10-14:1.

[0049] This invention provides a method for expressing exogenous genes loaded with PEI-functionalized carboxyl fluorescent quantum dots in eukaryotic cells. EPC cells (endothelial progenitor cells) were seeded in 12-well plates and cultured in M199 medium containing 10% FBS at 25°C for 24 hours. The medium was then replaced with M199 medium containing 5% FBS, and PEI-functionalized carboxyl fluorescent quantum dots loaded with pEGFPN1 plasmid (linking method as in S4) were added to the medium at a final concentration of 5 mg / L (based on plasmid concentration). The cells were observed under a fluorescence microscope after 48 hours. Figure 2 As shown. By Figure 2 It is known that PEI-functionalized carboxyl fluorescent quantum dots can deliver pEGFPN1 plasmid for expression in EPC cells.

[0050] In this embodiment of the invention, a method for preparing a carboxyl fluorescent quantum dot-loaded gene editing system is provided. The method includes the preparation of functionalized carboxyl fluorescent quantum dots and the ligation of the functionalized carboxyl fluorescent quantum dots with plasmid DNA.

[0051] The preparation of the functionalized carboxyl fluorescent quantum dots includes:

[0052] S1. Carboxyl fluorescent quantum dots with a carboxyl content of 6 μM / mL were dispersed in pure water at 0.05 μM, and N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added to a final concentration of 12 μM / mL. The mixture was stirred at room temperature for 2 h to activate the carboxyl groups on the surface of the carboxyl fluorescent quantum dots.

[0053] S2. Add 60 μM / mL branched polyethyleneimine to the carboxyl-activated carboxyl fluorescent quantum dot reaction system and stir at room temperature for 24 h;

[0054] S3. The reaction system was dialyzed using a 7000D dialysis bag to remove unreacted N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and branched polyethyleneimine. The resulting branched polyethyleneimine functionalized carboxyl fluorescent quantum dots are functionalized carboxyl fluorescent quantum dots. They do not aggregate within 90 days when stored at 4°C.

[0055] The connection between the functionalized carboxyl fluorescent quantum dots and plasmid DNA includes:

[0056] Plasmid DNA with a final concentration of 100 ng / μL was diluted in 100 μL of deionized water. 100-400 μL of functionalized carboxyl fluorescent quantum dots that had been sonicated (500 W, 3-5 s) were added to the solution. The mixture was then incubated at 150 rpm for 30 min at room temperature to obtain a carboxyl fluorescent quantum dot-loaded gene editing system, which was subsequently used for gene editing of fertilized eggs or cell transfection in aquatic animals.

[0057] This invention provides a method for digesting the eggshells of fertilized eggs from zebrafish, grass carp, rare gudgeon, largemouth bass, and oblique grouper, and for editing their genes using a PEI-functionalized carboxyl fluorescent quantum dot-loaded gene editing system, further illustrated with specific implementation examples.

[0058] Example 1

[0059] This embodiment provides zebrafish fertilized eggs obtained through artificial breeding in the laboratory of Northwest A&F University, and the zebrafish eggshells are digested according to the following method:

[0060] Method a-1: Digest with 0.50% papain for 2 min at 28℃, terminate digestion with 5% BSA, and wash 5 times with pure water;

[0061] Method a-2: Digest with 0.75% keratinase for 4 min at 28℃, terminate digestion with 5% BSA, and wash 5 times with pure water;

[0062] Method a-3: Digest with 1.00% chymotrypsin at 28℃ for 4 min, terminate digestion with 5% BSA, and wash 5 times with pure water;

[0063] To determine the efficiency of eggshell removal, zebrafish eggs with their shells removed were placed in a 25°C constant temperature incubator for incubation, and the survival rate after 6 hours was recorded.

[0064] The removal efficiency of zebrafish eggshells is shown in Table 1. It can be seen that the eggshell removal rates of methods a-1, a-2, and a-3 are 92.4%, 94.8%, and 96.2%, respectively.

[0065] Table 1. Zebrafish eggshell removal efficiency

[0066]

[0067] After removing the eggshell as described above, PEI-functionalized carboxyl fluorescent quantum dots loaded with the pEGFPN1 plasmid were added to the embryo hatching medium. Gene expression efficiency was statistically analyzed, and zebrafish that successfully expressed fluorescence were identified. Figure 3 As shown.

[0068] Example 2

[0069] This embodiment provides a method for obtaining rare gudgeon fertilized eggs through artificial spawning in the laboratory of Northwest A&F University, and digesting the eggshells using the following method.

[0070] Method b-1: Digest with 0.50% papain + 0.3% trypsin + 0.15% streptomycin at 25℃ for 6 min, terminate digestion with 10% BSA, and wash 5 times with pure water.

[0071] Method b-2: Digest with 2.00% papain + 0.6% streptomycin at 25℃ for 4 min, terminate digestion with 10% BSA, and wash 5 times with purified water.

[0072] Method b-3: Digest with 2.00% papain + 0.75% keratinase at 25℃ for 4 min, terminate digestion with 10% BSA, and wash 5 times with purified water.

[0073] The efficiency of eggshell removal was statistically analyzed, and the rare gudgeon with the eggshells removed were incubated in a 25℃ constant temperature incubator. The survival rate after 6 hours was then calculated.

[0074] The removal efficiency of rare gudgeon eggshells is shown in Table 2. It can be seen that the eggshell removal rates of methods b-1, b-2, and b-3 are 78.8%, 81.4%, and 84.4%, respectively.

[0075] Table 2. Removal efficiency of rare gudgeon eggshells

[0076]

[0077] After removing the eggshells as described above, PEI-functionalized carboxyl fluorescent quantum dots loaded with pEGFPN1 plasmid were added to the embryo hatching solution. Gene expression efficiency was statistically analyzed, and rare gudgeon that successfully expressed fluorescence was obtained.

[0078] Example 3

[0079] This embodiment provides grass carp fertilized eggs obtained through artificial spawning in the laboratory of Northwest A&F University, and the eggshell removal is performed according to the following method:

[0080] Method c-1: Digest with 0.75% papain + 0.25% trypsin + 0.10% streptomycin at 25℃ for 6 min, terminate digestion with 10% BSA, and wash 5 times with pure water.

[0081] Method c-2: Digest with 1.00% papain + 0.75% keratinase at 25℃ for 4 min, terminate digestion with 10% BSA, and wash 5 times with purified water.

[0082] Method c-3: Digest with 1.50% papain + 1.00% chymotrypsin at 25℃ for 4 min, terminate digestion with 10% BSA, and wash 5 times with purified water.

[0083] The efficiency of eggshell removal was statistically analyzed, and the grass carp with removed eggshells were incubated in a 25℃ constant temperature incubator. The survival rate after 6 hours was then calculated.

[0084] The removal efficiency of grass carp eggshells is shown in Table 3. It can be seen that the eggshell removal rates of methods c-1, c-2 and c-3 are 84.8%, 87.2% and 91.0%, respectively.

[0085] Table 3. Grass carp eggshell removal efficiency

[0086]

[0087] After removing the eggshells as described above, PEI-functionalized carboxyl fluorescent quantum dots loaded with pEGFPN1 plasmid were added to the embryo hatching solution. Gene expression efficiency was statistically analyzed, and grass carp that successfully expressed fluorescence were obtained.

[0088] Example 4

[0089] This embodiment provides a method for obtaining fertilized largemouth bass eggs through natural spawning, and removing the eggshells as follows:

[0090] Method d-1: Digest with 0.50% papain + 0.25% trypsin + 0.10% streptomycin at 25℃ for 6 min, terminate digestion with 10% BSA, and wash 5 times with pure water.

[0091] Method d-2: Digest with 2.00% papain + 0.5% keratinase for 5 min at 25℃, terminate digestion with 10% BSA, and wash 5 times with purified water.

[0092] Method d-3: Digest with 2.00% papain + 0.5% keratinase for 5 min at 25℃, terminate digestion with 10% BSA, and wash 5 times with purified water.

[0093] The efficiency of eggshell removal was statistically analyzed, and the largemouth bass eggs with the eggshells removed were incubated in a 25℃ constant temperature incubator. The survival rate after 6 hours was then calculated.

[0094] The shell removal efficiency of largemouth bass eggs is shown in Table 4. It can be seen that the shell removal rates of methods d-1, d-2 and d-3 are 79.2%, 83.2% and 93.4%, respectively.

[0095] Table 4. Eggshell Removal Efficiency of Largemouth Bass

[0096]

[0097] After removing the eggshells as described above, PEI-functionalized carboxyl fluorescent quantum dots loaded with pEGFPN1 plasmid were added to the embryo hatching solution. Gene expression efficiency was statistically analyzed, and largemouth bass that successfully expressed fluorescence were obtained.

[0098] Example 5

[0099] This embodiment provides a method for obtaining fertilized eggs of the oblique grouper through natural spawning, and removing the eggshell as follows.

[0100] Method e-1: Digest with 0.50% papain + 0.25% trypsin + 0.10% streptomycin at 25℃ for 5 min, terminate digestion with 10% BSA, and wash 5 times with 2.0% sea salt.

[0101] Method e-2: Digest with 0.50% papain + 0.20% chymotrypsin at 25℃ for 5 min, terminate digestion with 10% BSA, and wash 5 times with 2.0% sea salt.

[0102] Method e-3: Digest with 0.50% papain + 0.20% streptomycin + 0.25% chymotrypsin at 25℃ for 5 min, terminate digestion with 10% BSA, and wash 5 times with 2.0% sea salt.

[0103] The efficiency of eggshell removal was statistically analyzed, and the eggs of the grouper with the eggshells removed were incubated in a constant temperature incubator at 28℃. The survival rate after 6 hours was then calculated.

[0104] The removal efficiency of the eggshells of the oblique grouper is shown in Table 5. It can be seen that the eggshell removal rates of methods d-1, d-2 and d-3 are 77.6%, 78.8% and 83.4%, respectively.

[0105] Table 5. Eggshell removal efficiency of *Scutellaria edulis*

[0106]

[0107]

[0108] After removing the eggshells as described above, PEI-functionalized carboxyl fluorescent quantum dots loaded with pEGFPN1 plasmid were added to the embryo hatching solution. Gene expression efficiency was statistically analyzed, and fluorescently expressed grouper was obtained.

[0109] Example 6

[0110] This embodiment provides a gene editing method using a carboxyl fluorescent quantum dot-loaded gene editing system, as follows:

[0111] Method (1):

[0112] Prepare carboxylated fluorescent quantum dots (carboxylated fluorescent quantum dot-loaded gene editing system) in advance using the method described above;

[0113] Then, zebrafish fertilized eggs are collected through artificial breeding or natural spawning. The eggs are washed several times to remove impurities, algae, bacteria, etc., adsorbed on the egg surface. The fertilized eggs are placed in a suitable enzyme system, and then gently agitated and stirred continuously using a dropper. After digestion, the enzyme solution is discarded, and a BSA solution of the appropriate concentration is added to terminate the enzyme digestion (as described in Example 1). The eggs are washed several times, and then the shelled eggs are carefully added to a solution containing carboxyl fluorescent quantum dots loaded with plasmids. After immersion for 2 hours, the embryos are washed and then incubated to complete the gene editing process.

[0114] Gene expression efficiency was statistically analyzed using fluorescence microscopy, and the expression efficiency is shown in Table 6.

[0115] After removing the eggshell as described above, PEI-functionalized carboxyl fluorescent quantum dots loaded with the pEGFPN1 plasmid were added to the embryo hatching medium. Gene expression efficiency was statistically analyzed, and zebrafish that successfully expressed green fluorescent protein were identified. Figure 3 As shown, Bright refers to the image under the white light channel, and GFP refers to the image under the GFP channel. (From...) Figure 3 It can be seen that PEI-functionalized carboxyl fluorescent quantum dots successfully delivered pEGFPN1 plasmid for expression in zebrafish embryos.

[0116] The tyr gene was knocked out in molted zebrafish eggs using a carboxyl fluorescent quantum dot-loaded gene editing system, and the ligation method was the same as the above-mentioned delivery of the pEGFPN1 plasmid for verification. Pigment accumulation in zebrafish 48 hours post-fertilization before and after knockout was as follows: Figure 4 As shown, the body color accumulation of the carboxyl fluorescent quantum dot group is significantly less than that of the control group, which is most obvious in the eye area.

[0117] The sequences of the zebrafish tyr gene before and after knockout were compared using Sanger sequencing, and the peak diagram is shown below. Figure 5 As shown, a significant upregulation of heterogeneous peaks near PAM at gRNA was observed in the carboxyl fluorescent quantum dot group, indicating that the gene editing method of the carboxyl fluorescent quantum dot loaded gene editing system in zebrafish is feasible.

[0118] Method (2):

[0119] Prepare carboxylated fluorescent quantum dots (carboxylated fluorescent quantum dot-loaded gene editing system) in advance using the method described above;

[0120] Then, through artificial breeding or natural spawning, rare gudgeon fertilized eggs are collected. The eggs are washed several times to remove impurities, algae, bacteria, etc., adsorbed on the egg surface. The fertilized eggs are placed in a suitable enzyme system, and then gently blown and stirred continuously with a dropper. After digestion, the enzyme solution is discarded, and a BSA solution of the appropriate concentration is added to terminate the enzyme digestion (as described in the steps of Example 2). The eggs are washed several times, and then the shelled fish eggs are carefully added to a solution containing carboxyl fluorescent quantum dots loaded with plasmids. After soaking for 2 hours, the embryos are washed and then incubated to complete the gene editing process.

[0121] The effect of carboxyl fluorescent quantum dot loading on pEGFPN1 plasmid delivery was observed by fluorescence microscopy. The GFP gene expression efficiency at 24h and 48h is shown in Table 6.

[0122] After removing the eggshells as described above, PEI-functionalized carboxyl fluorescent quantum dots loaded with the pEGFPN1 plasmid were added to the embryo hatching medium, and gene expression efficiency was statistically analyzed. Similarly, rare gudgeon expressing green fluorescent protein was successfully obtained.

[0123] The tyr gene was knocked out in molted rare gudgeon eggs using a carboxylated fluorescent quantum dot-loaded gene editing system, and the ligation method was the same as the above-mentioned delivery of the pEGFPN1 plasmid for verification. By observing the pigment accumulation of rare gudgeon 48 hours after fertilization before and after knockout, it was found that the body color accumulation in the carboxylated fluorescent quantum dot group was significantly less than that in the control group.

[0124] By comparing the sequences of the rare gudgeon's tyr gene before and after knockout using Sanger sequencing, it was found that the carboxyl fluorescent quantum dot group showed a significant upregulation of heterogeneous peaks near PAM at the gRNA, indicating that the gene editing method of the carboxyl fluorescent quantum dot loaded gene editing system in rare gudgeon is feasible.

[0125] Method (3):

[0126] Prepare carboxylated fluorescent quantum dots (carboxylated fluorescent quantum dot-loaded gene editing system) in advance using the method described above;

[0127] Then, grass carp fertilized eggs are collected through artificial breeding or natural spawning. The eggs are washed several times to remove impurities, algae, bacteria, etc., adsorbed on the egg surface. The fertilized eggs are placed in a suitable enzyme system, and then gently blown and stirred continuously with a dropper. After digestion, the enzyme solution is discarded and a BSA solution of the appropriate concentration is added to terminate the enzyme digestion (as described in the steps of Example 3). The eggs are washed several times, and then the shelled fish eggs are carefully added to a solution containing carboxyl fluorescent quantum dots loaded with plasmids. After soaking for 2 hours, the embryos are washed and then incubated to complete the gene editing process.

[0128] The effect of carboxyl fluorescent quantum dot loading on pEGFPN1 plasmid delivery was observed by fluorescence microscopy. The GFP gene expression efficiency at 24h and 48h is shown in Table 6.

[0129] After removing the eggshells as described above, PEI-functionalized carboxyl fluorescent quantum dots loaded with the pEGFPN1 plasmid were added to the embryo hatching medium, and gene expression efficiency was statistically analyzed. Similarly, grass carp expressing green fluorescent protein were successfully obtained.

[0130] The tyr gene was knocked out in molting grass carp eggs using a carboxylated fluorescent quantum dot-loaded gene editing system, and the ligation method was the same as the above-mentioned delivery of the pEGFPN1 plasmid for verification. By observing the pigment accumulation of grass carp before and after fertilization at 48 hours, it was found that the body color accumulation in the carboxylated fluorescent quantum dot group was significantly less than that in the control group, which was most obvious in the eyes.

[0131] By comparing the sequences of the grass carp tyr gene before and after knockout using Sanger sequencing, it was found that the carboxyl fluorescent quantum dot group showed a significant upregulation of heterogeneous peaks near PAM at the gRNA, indicating that the gene editing method of the carboxyl fluorescent quantum dot loaded gene editing system in grass carp is feasible.

[0132] Method (4):

[0133] Prepare carboxylated fluorescent quantum dots (carboxylated fluorescent quantum dot-loaded gene editing system) in advance using the method described above;

[0134] Then, through artificial breeding or natural spawning, fertilized eggs of largemouth bass are collected. The eggs are washed several times to remove impurities, algae, bacteria, etc., adsorbed on the egg surface. The fertilized eggs are placed in a suitable enzyme system, and then gently blown and stirred continuously with a dropper. After digestion, the enzyme solution is discarded, and a BSA solution of the appropriate concentration is added to terminate the enzyme digestion (as described in the steps of Example 4). The eggs are washed several times, and then the shelled fish eggs are carefully added to a solution containing carboxyl fluorescent quantum dots loaded with plasmids. After soaking for 2 hours, the embryos are washed and then incubated to complete the gene editing process.

[0135] The effect of carboxyl fluorescent quantum dot loading on pEGFPN1 plasmid delivery was observed by fluorescence microscopy. The GFP gene expression efficiency at 24h and 48h is shown in Table 6.

[0136] After removing the eggshells as described above, PEI-functionalized carboxyl fluorescent quantum dots carrying the GFP gene plasmid were added to the embryo hatching medium, and the gene expression efficiency was statistically analyzed. Similarly, largemouth bass expressing green fluorescent protein were successfully obtained.

[0137] The tyr gene was knocked out in molted largemouth bass eggs using a carboxylated fluorescent quantum dot-loaded gene editing system, and the ligation method was the same as the above-mentioned delivery of pEGFPN1 plasmid for verification. By observing the pigment accumulation of grass carp before and after knockout 48 hours after fertilization, it was found that the body color accumulation in the carboxylated fluorescent quantum dot group was significantly less than that in the control group.

[0138] By comparing the sequences of the tyr gene in largemouth bass before and after knockout using Sanger sequencing, it was found that the carboxyl fluorescent quantum dot group showed a significant upregulation of heterogeneous peaks near PAM at the gRNA, indicating that the gene editing method of the carboxyl fluorescent quantum dot loaded gene editing system in largemouth bass is feasible.

[0139] Method (5):

[0140] Prepare carboxylated fluorescent quantum dots (carboxylated fluorescent quantum dot-loaded gene editing system) in advance using the method described above;

[0141] Then, through artificial breeding or natural spawning, fertilized eggs of the oblique grouper are collected. The eggs are washed several times to remove impurities, algae, bacteria, etc., adsorbed on the egg surface. The fertilized eggs are placed in a suitable enzyme system, and then gently blown and stirred continuously with a dropper. After digestion, the enzyme solution is discarded, and a BSA solution of the appropriate concentration is added to terminate the enzyme digestion (as described in the steps of Example 5). The eggs are washed several times, and then the shelled fish eggs are carefully added to a solution containing carboxyl fluorescent quantum dots loaded with plasmids. After soaking for 2 hours, the embryos are washed and then incubated to complete the gene editing process.

[0142] The effect of carboxyl fluorescent quantum dot loading on pEGFPN1 plasmid delivery was observed by fluorescence microscopy. The GFP gene expression efficiency at 24h and 48h is shown in Table 6.

[0143] After removing the eggshells as described above, PEI-functionalized carboxyl fluorescent quantum dots loaded with the pEGFPN1 plasmid were added to the embryo hatching medium, and gene expression efficiency was statistically analyzed. Similarly, grouper expressing green fluorescent protein were successfully obtained.

[0144] The tyr gene was knocked out in molted grouper eggs using a carboxyl fluorescent quantum dot-loaded gene editing system, and the ligation method was the same as that described above for delivery with the pEGFPN1 plasmid. By observing the pigment accumulation of grouper before and after knockout at 48 h post-fertilization, it was found that the body color accumulation in the carboxyl fluorescent quantum dot group was significantly less than that in the control group, which was most obvious in the eyes.

[0145] By comparing the sequences of the tyr gene in *Sinocyclocheilus scoparia* before and after knockout using Sanger sequencing, it was found that the carboxyl fluorescent quantum dot group showed a significant upregulation of heterogeneous peaks near PAM at the gRNA, indicating that the gene editing method of the carboxyl fluorescent quantum dot loaded gene editing system in *Sinocyclocheilus scoparia* is feasible.

[0146] Table 6. Expression rate of exogenous genes delivered by carboxyl fluorescent quantum dots in fish eggs.

[0147]

[0148] As shown in Table 6, PEI-functionalized carboxyl fluorescent quantum dots can deliver exogenous genes in zebrafish, rare gudgeon, grass carp, largemouth bass, and oblique grouper.

[0149] It should be understood that the disclosed invention is not limited to the specific methods, schemes, and substances described, as these are all subject to variation. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims.

[0150] Those skilled in the art will also recognize, or be able to identify, many equivalents of the specific embodiments of the invention described herein using no more than conventional experiments. These equivalents are also included in the appended claims.

Claims

1. The application of a carboxyl fluorescent quantum dot-loaded gene editing system in gene editing of fertilized eggs of aquatic animals, characterized in that, The shell of the fertilized egg was digested by an enzyme preparation; The aquatic animals include zebrafish, rare gudgeon, grass carp, largemouth bass, and oblique grouper; the enzyme preparation is selected from one or more of trypsin, streptomycin, papain, keratinase, and chymotrypsin. The carboxylated fluorescent quantum dot-loaded gene editing system was delivered to aquatic animal fertilized eggs by immersion in the system for 0.5-2 hours after the eggshells were removed. The application concentration of the carboxyl fluorescent quantum dot loaded gene editing system is 1-50 mg / L; The preparation method of the carboxyl fluorescent quantum dot-loaded gene editing system includes the preparation of functionalized carboxyl fluorescent quantum dots and the ligation of functionalized carboxyl fluorescent quantum dots with plasmid DNA; wherein... The preparation of the functionalized carboxyl fluorescent quantum dots includes: S1. Disperse carboxyl fluorescent quantum dots in pure water, add N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and stir the reaction at room temperature to activate the carboxyl groups on the surface of the carboxyl fluorescent quantum dots; S2. Branched polyethyleneimine was added to the carboxyl-activated carboxyl fluorescent quantum dot reaction system, and the reaction was stirred at room temperature; S3. The reaction system was dialyzed using a 7000 D dialysis bag to remove unreacted N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and branched polyethyleneimine. The resulting branched polyethyleneimine functionalized carboxyl fluorescent quantum dots are functionalized carboxyl fluorescent quantum dots, which were stored at 4℃. The connection between the functionalized carboxyl fluorescent quantum dots and plasmid DNA includes: Plasmid DNA was diluted in deionized water, and functionalized carboxyl fluorescent quantum dots that had been sonicated were added to the solution. The mixture was then incubated at room temperature with shaking to obtain a carboxyl fluorescent quantum dot-loaded gene editing system, which was subsequently used for gene editing of fertilized eggs of aquatic animals or cell transfection. The ratio of the number of nitrogen atoms N in the branched polyethyleneimine to the number of negative charges P in the plasmid DNA is in the range of 10-14:1; The particle size of the carboxyl fluorescent quantum dots is 1-10 nm; the molecular weight of the branched polyethyleneimine is 900 g / M; and the pH of the branched polyethyleneimine is 7.

2. The application according to claim 1, characterized in that, The preparation of the functionalized carboxyl fluorescent quantum dots includes: S1. Carboxyl fluorescent quantum dots with a carboxyl content of 6 μM / mL were dispersed in pure water at 0.05 μM, and N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added to a final concentration of 12 μM / mL. The mixture was stirred at room temperature for 2 h to activate the carboxyl groups on the surface of the carboxyl fluorescent quantum dots. S2. Add 60 μM / mL of branched polyethyleneimine to the carboxyl-activated carboxyl fluorescent quantum dot reaction system and stir at room temperature for 24 h; S3. The reaction system was dialyzed using a 7000 D dialysis bag to remove unreacted N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and branched polyethyleneimine. The resulting branched polyethyleneimine functionalized carboxyl fluorescent quantum dots are functionalized carboxyl fluorescent quantum dots. They do not aggregate within 90 days when stored at 4°C. The connection between the functionalized carboxyl fluorescent quantum dots and plasmid DNA includes: Plasmid DNA with a final concentration of 100 ng / μL was diluted in 100 μL of deionized water, and 100-400 μL of sonicated functionalized carboxyl fluorescent quantum dots were added. The mixture was then incubated at 150 rpm for 30 min at room temperature to obtain a carboxyl fluorescent quantum dot-loaded gene editing system, which was subsequently used for gene editing of fertilized eggs or cell transfection in aquatic animals.

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