Carboxyl fluorescent quantum dot loaded gene editing system and application thereof in aquatic animal gene editing

Abstract

The invention relates to the technical field of aquaculture, in particular to a carboxyl fluorescent quantum dot loaded gene editing system and application thereof in aquatic animal gene editing. The invention provides a preparation method of a carboxyl fluorescent quantum dot loaded gene editing system, which comprises preparation of functionalized carboxyl fluorescent quantum dots and connection of the functionalized carboxyl fluorescent quantum dots and plasmid DNA to obtain the carboxyl fluorescent quantum dot loaded gene editing system, and the carboxyl fluorescent quantum dot loaded gene editing system can be subsequently used for aquatic animal fertilized egg gene editing or cell transfection. According to the method, safe and efficient egg shell digestive enzymes and nano materials are used for gene editing breeding of aquatic animals.

Description

Technical Field

[0001] The present invention relates to the technical field of aquaculture, and in particular to a carboxyl fluorescent quantum dot-loaded gene editing system and application thereof in gene editing of aquatic animals. Background Art

[0002] In recent years, the aquaculture industry has flourished, but the breeding of aquatic animals faces many challenges. Grass carp (Ctenopharyngodon idellus), largemouth bass (Micropterus salmoides), and coiate grouper (Epinephelus coioides) are important economic aquatic animals. At the same time, zebrafish (Danio rerio) and rare goby (Gobiocypris rarus) are important model animals and are widely used as research subjects in various experiments. Aquatic animal gene editing breeding technology is of great significance to the development of the aquaculture industry. It is an important means to improve the quality of aquatic seed industry. It is of great significance to develop new gene editing methods for important economic animals and model animals. Therefore, the development of an efficient and large-scale gene editing delivery platform for aquatic animals is of great significance to the development of improved aquatic seed industry.

[0003] Traditional microinjection for gene editing is inefficient, limited by equipment, and easily causes embryo rupture, which seriously affects the hatching rate of aquatic animal embryos. Therefore, it is crucial to develop a new type of large-scale and efficient gene editing system. The eggshell of aquatic animal fertilized eggs is an important structure for maintaining the stability of the embryonic environment and is of great significance to embryonic development. Nanomaterials have been widely used in drug delivery, but their application in gene editing delivery in aquatic animals has not yet been realized. The presence of the eggshell will hinder substances such as nanomaterials or biomacromolecules from entering the embryo. Destroying the eggshell of aquatic animal embryos is of great significance for the nanomaterial delivery plasmid system.

[0004] Therefore, it is urgent to find safe and efficient eggshell digestive enzymes and nanomaterials for gene editing breeding of aquatic animals. Summary of the invention

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

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

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

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

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

[0010] S2. adding branched polyethyleneimine to the carboxyl-activated carboxyl fluorescent quantum dot reaction system and stirring the reaction 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, and the obtained branched polyethyleneimine functionalized carboxyl fluorescent quantum dots, namely functionalized carboxyl fluorescent quantum dots, were stored at 4°C;

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

[0013] The plasmid DNA is diluted in deionized water, to which the functionalized carboxyl fluorescent quantum dots treated with ultrasound are added, and the mixture is incubated with shaking at room temperature to obtain a carboxyl fluorescent quantum dot-loaded gene editing system, which is subsequently used for gene editing of fertilized eggs of aquatic animals or cell transfection.

[0014] Preferably,

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

[0016] S1. Disperse carboxyl fluorescent quantum dots with a carboxyl content of 6 μM / mL in pure water at 0.05 μM, add N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride at a final concentration of 12 μM / mL, and stir 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 of branched polyethyleneimine to the carboxyl-activated carboxyl fluorescent quantum dot reaction system and stir the reaction at room temperature for 24 h;

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

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

[0020] The plasmid DNA with a final concentration of 100 ng / μL was diluted in 100 μL of deionized water, and 100-400 μL of functionalized carboxyl fluorescent quantum dots after ultrasonic treatment (power 500 w, time 3-5 s) were added thereto. The mixture was incubated at room temperature with shaking at 150 rpm for 30 min 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.

[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] In a second aspect, a carboxyl fluorescent quantum dot-loaded gene editing system is provided, which is prepared by the preparation method described in the present invention.

[0024] In a third aspect, there is provided an application of the carboxyl fluorescent quantum dot-loaded gene editing system of the present invention 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 goby, grass carp, largemouth bass and coioidal grouper; the enzyme preparation is selected from one or more of trypsin, pronase, papain, keratinase and chymotrypsin.

[0026] Preferably, the carboxyl fluorescent quantum dot-loaded gene editing system is delivered to the fertilized eggs of aquatic animals by immersion, and the fertilized eggs of aquatic animals with the egg shells removed 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] In a fourth aspect, the application of the carboxyl fluorescent quantum dot-loaded gene editing system described in the present invention in an aquatic animal cell line is provided.

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

[0030] Fluorescent quantum dots are a new type of nanoparticles, which are semiconductor nanoparticles composed of group 0 elements that can receive excitation light to produce fluorescence. They have unique quantum size effects and surface effects, and have the advantages of easy surface modification, excellent optical properties, and good biosafety and biocompatibility. The fluorescent quantum dots used in the present invention have extremely high potential and application value in delivering nucleic acids for gene editing in aquatic animals, and provide new strategies and methods for gene editing in aquatic animals.

[0031] The method for digesting the eggshell of aquatic animal fertilized egg with an enzyme preparation provided by the present invention comprises the combined use of papain, keratinase, chymotrypsin, trypsin and pronase, which removes the first obstacle for the nanomaterial delivery gene editing system.

[0032] The invention provides the activation of fluorescent quantum dots by N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride to obtain carboxyl-activated carboxyl fluorescent quantum dots, making them nanoparticles capable of loading gene editing systems. Furthermore, the invention provides PEI (polyethyleneimine) functional modification of fluorescent quantum dots to make them nanoparticles capable of better loading gene editing systems, and constructs nanocomplexes for gene editing of fertilized eggs of aquatic animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of carboxyl fluorescent quantum dots.

[0034] Figure 2 Fluorescence microscopy was used to observe the expression of exogenous GFP gene in EPC cells by delivering carboxyl fluorescent quantum dots loaded gene editing system (plasmid loaded with GFP gene).

[0035] Figure 3 The carboxyl fluorescent quantum dot-loaded gene editing system (plasmid loaded with GFP gene) is used to deliver exogenous GFP gene for expression in zebrafish eggs.

[0036] Figure 4 The carboxyl fluorescent quantum dot-loaded gene editing system (plasmid loaded with the tyr gene) was used to deliver the tyr gene, causing the zebrafish body color to change.

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

[0038] in, Figure 2-Figure 5 The control group refers to the group without carboxyl fluorescent quantum dots. DETAILED DESCRIPTION

[0039] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

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

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

[0042] S1. Disperse carboxyl fluorescent quantum dots (carboxyl content of 6 μM / mL) in pure water at 0.05 μM, add NHS (N-hydroxysuccinimide) and EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride) at a final concentration of 12 μM / mL, and stir 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 the reaction at room temperature for 24 h;

[0044] Wherein, 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.

[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 were stored at 4°C and would not aggregate within 90 days. Figure 1 shown.

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

[0047] S4. Dilute the plasmid DNA in 100 μL deionized water to a final concentration of 100 ng / μL, add 100-400 μL of ultrasonically treated PEI functionalized carboxyl fluorescent quantum dots, 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] The embodiment of the present 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 plated in a 12-well plate and cultured in a 25°C constant temperature incubator for 24 hours with M199 medium containing 10% FBS; the medium was replaced with M199 medium containing 5% FBS, and PEI functionalized carboxyl fluorescent quantum dots loaded with pEGFPN1 plasmid at a final concentration of 5 mg / L (in terms of plasmid concentration) were added to the medium (the connection method is the same as S4). Observe under a fluorescence microscope for 48 hours, as shown in FIG. Figure 2 As shown. Figure 2 It can be seen that PEI-functionalized carboxyl fluorescent quantum dots can deliver pEGFPN1 plasmid for expression in EPC cells.

[0050] In an embodiment of the present invention, a method for preparing a carboxyl fluorescent quantum dot-loaded gene editing system is provided, the preparation method comprising the preparation of functionalized carboxyl fluorescent quantum dots and the connection of the functionalized carboxyl fluorescent quantum dots to plasmid DNA; wherein,

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

[0052] S1. Disperse carboxyl fluorescent quantum dots with a carboxyl content of 6 μM / mL in pure water at 0.05 μM, add N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride at a final concentration of 12 μM / mL, and stir 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 of branched polyethyleneimine to the carboxyl-activated carboxyl fluorescent quantum dot reaction system and stir the reaction at room temperature for 24 h;

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

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

[0056] The plasmid DNA with a final concentration of 100 ng / μL was diluted in 100 μL of deionized water, and 100-400 μL of functionalized carboxyl fluorescent quantum dots after ultrasonic treatment (power 500 w, time 3-5 s) were added thereto. The mixture was incubated at room temperature with shaking at 150 rpm for 30 min 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.

[0057] The embodiments of the present invention provide a method for digesting the egg shells of fertilized eggs of zebrafish, grass carp, rare goby, largemouth seabass, and coioidal grouper, and use a PEI-functionalized carboxyl fluorescent quantum dot-loaded gene editing system to edit their genes, which is further illustrated in conjunction with specific implementation cases.

[0058] Example 1

[0059] This example provides zebrafish fertilized eggs obtained by artificial breeding in the laboratory of Northwest Agriculture and Forestry University, and zebrafish egg shell digestion is performed according to the following method:

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

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

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

[0063] The eggshell removal efficiency was calculated, and the zebrafish eggs without eggshells were placed in a 25°C constant temperature incubator for incubation, and the 6-h survival rate was calculated.

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

[0065] Table 1 Zebrafish egg shell removal efficiency

[0066]

[0067] After removing the egg shell as described above, PEI-functionalized carboxyl fluorescent quantum dots loaded with pEGFPN1 plasmid were added to the embryo incubation medium, and the gene expression efficiency was counted. The zebrafish that successfully expressed fluorescent Figure 3 shown.

[0068] Example 2

[0069] This example provides methods of obtaining fertilized eggs of rare goby carp by artificially inducing spawning of rare goby carp in the laboratory of Northwest Agriculture and Forestry University, and digesting the egg shells using the following method.

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

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

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

[0073] The egg shell removal efficiency was calculated, and the rare goby with the egg shell removed was incubated in a constant temperature incubator at 25°C, and the 6-hour survival rate was calculated.

[0074] The removal efficiency of egg shell of rare crucian carp is shown in Table 2. It can be seen that the egg shell 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 egg shell of rare crucian carp

[0076]

[0077] After the egg shell was removed according to the above method, PEI functionalized carboxyl fluorescent quantum dots loaded with pEGFPN1 plasmid were added to the embryo hatching solution, and the gene expression efficiency was counted, and the rare goby that successfully expressed fluorescence was also obtained.

[0078] Example 3

[0079] This embodiment provides that fertilized grass carp eggs are obtained by artificial induced spawning in the laboratory of Northwest Agriculture and Forestry University, and the egg shells are removed according to the following method:

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

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

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

[0083] The eggshell removal efficiency was calculated, and the grass carp with the eggshell removed were incubated in a constant temperature incubator at 25°C, and the 6-hour survival rate was calculated.

[0084] The removal efficiency of grass carp egg shells is shown in Table 3. It can be seen that the egg shell 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 egg shell removal efficiency

[0086]

[0087] After removing the egg shell according to the above method, PEI functionalized carboxyl fluorescent quantum dots loaded with pEGFPN1 plasmid were added to the embryo hatching solution, and the gene expression efficiency was counted, and grass carp that successfully expressed fluorescence was also obtained.

[0088] Example 4

[0089] This embodiment provides fertilized eggs of largemouth bass obtained by natural spawning, and the egg shells are removed according to the following method:

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

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

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

[0093] The eggshell removal efficiency was calculated, and the largemouth bass eggs without eggshells were incubated in a constant temperature incubator at 25°C, and the 6-hour survival rate was calculated.

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

[0095] Table 4 Largemouth bass egg shell removal efficiency

[0096]

[0097] After the egg shell was removed according to the above method, PEI functionalized carboxyl fluorescent quantum dots loaded with pEGFPN1 plasmid were added to the embryo hatching solution, and the gene expression efficiency was counted, and largemouth bass that successfully expressed fluorescence were also obtained.

[0098] Example 5

[0099] This embodiment provides fertilized eggs of Epinephelus coioides obtained by natural spawning, and the egg shells are removed according to the following method.

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

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

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

[0103] The eggshell removal efficiency was calculated, and the eggs of Epinephelus coioides with the eggshell removed were incubated in a constant temperature incubator at 28°C, and the 6-h survival rate was calculated.

[0104] The removal efficiency of egg shells of Epinephelus coioides is shown in Table 5. It can be seen that the egg shell removal rates of methods d-1, d-2, and d-3 are 77.6%, 78.8%, and 83.4%, respectively.

[0105] Table 5 Removal efficiency of egg shell of Epinephelus coioides

[0106]

[0107]

[0108] After removing the egg shell according to the above method, PEI functionalized carboxyl fluorescent quantum dots loaded with pEGFPN1 plasmid were added to the embryo hatching medium, and the gene expression efficiency was counted, and the coioidal grouper that successfully expressed fluorescence was also obtained.

[0109] Example 6

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

[0111] Method (1):

[0112] Prepare the carboxyl fluorescent quantum dots loaded with plasmids in advance according to the above method (carboxyl fluorescent quantum dots loaded gene editing system);

[0113] Then artificially propagate or spawn naturally, and collect zebrafish fertilized eggs. Wash the eggs several times to remove impurities, algae, bacteria, etc. adsorbed on the egg surface. The fertilized eggs are placed in the corresponding enzyme system, and then gently blown and stirred with a rubber-tipped dropper. After digestion, the enzyme solution is discarded and a BSA solution of corresponding concentration is added to terminate the digestion of the enzyme (as described in the steps of Example 1). Wash the eggs several times, then carefully add the fish eggs with the egg shells removed to the solution containing the carboxyl fluorescent quantum dots loaded with plasmids, wash the embryos after immersion for 2 hours, and then hatch them to complete the gene editing process.

[0114] The gene expression efficiency was statistically analyzed by fluorescence microscopy, and the expression efficiency is shown in Table 6.

[0115] After removing the egg shell as described above, PEI functionalized carboxyl fluorescent quantum dots loaded with pEGFPN1 plasmid were added to the embryo incubation medium, and the gene expression efficiency was counted. The zebrafish that successfully expressed green fluorescent protein were as follows: Figure 3 As shown in the figure, Bright refers to the photo taken under the white light channel, and GFP refers to the photo taken under the GFP channel. 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 shelled zebrafish eggs using the carboxyl fluorescent quantum dot-loaded gene editing system. The connection method was the same as the above-mentioned pEGFPN1 plasmid delivery verification. The pigment accumulation of zebrafish before and after the knockout at 48 hours after fertilization is shown in Figure 2. Figure 4 As shown, it can be seen that the body color accumulation of the carboxyl fluorescent quantum dots group is significantly less than that of the control group, which is most obvious in the eyes.

[0117] The sequences of zebrafish tyr gene before and after knockout were compared by Sanger sequencing. The peak diagram is shown in Figure 5 As shown, it can be seen that the carboxyl fluorescent quantum dot group showed an obvious upregulation of the mixed peak near the PAM of the gRNA, 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 the carboxyl fluorescent quantum dots loaded with plasmids in advance according to the above method (carboxyl fluorescent quantum dots loaded gene editing system);

[0120] Then artificially propagate or spawn naturally, and collect the fertilized eggs of rare crucian carp. Wash the eggs several times to remove impurities, algae, bacteria, etc. adsorbed on the surface of the eggs. The fertilized eggs are placed in the corresponding enzyme system, and then gently blown and stirred with a rubber-tipped dropper. After digestion, the enzyme solution is discarded and the BSA solution of the corresponding concentration is added to terminate the digestion of the enzyme (as described in the steps in Example 2). Wash the eggs several times, then carefully add the fish eggs with the egg shells removed to the solution containing the carboxyl fluorescent quantum dots loaded with plasmids, wash the embryos after immersion for 2 hours, and then hatch them to complete the gene editing process.

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

[0122] After removing the egg shell according to the above method, PEI functionalized carboxyl fluorescent quantum dots loaded with pEGFPN1 plasmid were added to the embryo hatching solution to calculate the gene expression efficiency. Similarly, rare goby expressing green fluorescent protein was successfully obtained.

[0123] The tyr gene was knocked out in shelled rare goby carp eggs using the carboxyl fluorescent quantum dot-loaded gene editing system, and the connection method was the same as the above-mentioned pEGFPN1 plasmid delivery verification. By observing the pigment accumulation of rare goby carp before and after knockout at 48 hours after fertilization, it can be seen that the body color accumulation of the carboxyl fluorescent quantum dot group is significantly less than that of the control group.

[0124] The sequences of the tyr gene of the rare goby before and after knockout were compared by Sanger sequencing, and it was found that the carboxyl fluorescent quantum dot group showed an obvious upregulation of the hybrid peak near the PAM of the gRNA, indicating that the gene editing method of the carboxyl fluorescent quantum dot-loaded gene editing system in the rare goby is feasible.

[0125] Method (3):

[0126] Prepare the carboxyl fluorescent quantum dots loaded with plasmids in advance according to the above method (carboxyl fluorescent quantum dots loaded gene editing system);

[0127] Then artificial breeding or natural spawning, collect grass carp fertilized eggs. Wash the eggs several times to remove impurities, algae, bacteria, etc. adsorbed on the egg surface. The fertilized eggs are placed in the corresponding enzyme system, and then gently blown and stirred with a rubber-tipped dropper. After digestion, the enzyme solution is discarded and the corresponding concentration of BSA solution is added to terminate the digestion of the enzyme (as described in the steps in Example 3). Wash the eggs several times, then carefully add the fish eggs with the egg shells removed to the solution containing the carboxyl fluorescent quantum dots loaded with plasmids, wash the embryos after immersion for 2 hours, and then hatch them to complete the gene editing process.

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

[0129] After removing the egg shell according to the above method, PEI functionalized carboxyl fluorescent quantum dots loaded with pEGFPN1 plasmid were added to the embryo hatching solution to calculate the gene expression efficiency. Similarly, grass carp expressing green fluorescent protein was successfully obtained.

[0130] The tyr gene was knocked out in shelled grass carp eggs using the carboxyl fluorescent quantum dot-loaded gene editing system, and the connection method was the same as the above-mentioned pEGFPN1 plasmid delivery verification. By observing the pigment accumulation of grass carp before and after knockout 48 hours after fertilization, it can be seen that 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 eyes.

[0131] The sequences of the grass carp tyr gene before and after knockout were compared by Sanger sequencing, and it was found that the carboxyl fluorescent quantum dot group showed an obvious upregulation of the hybrid peak near the PAM of 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 the carboxyl fluorescent quantum dots loaded with plasmids in advance according to the above method (carboxyl fluorescent quantum dots loaded gene editing system);

[0134] Then artificially propagate or spawn naturally, and collect the fertilized eggs of largemouth bass. Wash the eggs several times to remove impurities, algae, bacteria, etc. adsorbed on the surface of the eggs. The fertilized eggs are placed in the corresponding enzyme system, and then gently blown and stirred with a rubber-tipped dropper. After digestion, the enzyme solution is discarded and the BSA solution of the corresponding concentration is added to terminate the digestion of the enzyme (as described in the steps of Example 4). Wash the eggs several times, then carefully add the fish eggs with the egg shells removed to the solution containing the carboxyl fluorescent quantum dots loaded with plasmids, wash the embryos after immersion for 2 hours, and then hatch them to complete the gene editing process.

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

[0136] After the egg shell was removed according to the above method, PEI functionalized carboxyl fluorescent quantum dots loaded with the plasmid of GFP gene were added to the embryo hatching solution, and the gene expression efficiency was counted. Similarly, largemouth bass expressing green fluorescent protein were successfully obtained.

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

[0138] The sequences of the tyr gene of largemouth bass before and after knockout were compared by Sanger sequencing, and it was found that the carboxyl fluorescent quantum dot group showed an obvious upregulation of the mixed peak near the PAM of 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 the carboxyl fluorescent quantum dots loaded with plasmids in advance according to the above method (carboxyl fluorescent quantum dots loaded gene editing system);

[0141] Then artificially propagate or spawn naturally, and collect the fertilized eggs of Epinephelus coioides. Wash the eggs several times to remove impurities, algae, bacteria, etc. adsorbed on the surface of the eggs. The fertilized eggs are placed in the corresponding enzyme system, and then gently blown and stirred with a rubber-tipped dropper. After digestion, the enzyme solution is discarded and a BSA solution of the corresponding concentration is added to terminate the digestion of the enzyme (as described in the steps of Example 5). Wash the eggs several times, then carefully add the fish eggs with the egg shells removed to the solution containing the carboxyl fluorescent quantum dots loaded with plasmids, wash the embryos after immersion for 2 hours, and then hatch them to complete the gene editing process.

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

[0143] After removing the egg shell according to the above method, PEI functionalized carboxyl fluorescent quantum dots loaded with pEGFPN1 plasmid were added to the embryo hatching solution to calculate the gene expression efficiency. Similarly, the coioidal grouper expressing green fluorescent protein was successfully obtained.

[0144] The tyr gene was knocked out in shelled grouper eggs using the carboxyl fluorescent quantum dot-loaded gene editing system, and the connection method was the same as the above-mentioned pEGFPN1 plasmid delivery verification. By observing the pigment accumulation of grouper before and after knockout 48 hours after fertilization, it can be seen that 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 eyes.

[0145] The sequences of the tyr gene of Epinephelus coioides before and after knockout were compared by Sanger sequencing, and it was found that the carboxyl fluorescent quantum dot group showed an obvious upregulation of the hybrid peak near the PAM of the gRNA, indicating that the gene editing method of the carboxyl fluorescent quantum dot-loaded gene editing system in Epinephelus coioides 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 for expression in zebrafish, rare goby, grass carp, largemouth bass and coioidal grouper.

[0149] It should be understood that the disclosed invention is not limited only to the specific method, scheme and material of description, because these all can change.It should also be understood that the terminology used herein is only for the purpose of describing specific embodiment scheme, rather than being intended to limit the scope of the present invention, and the scope of the present invention is only limited to the appended claims.

[0150] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also intended to be encompassed by the appended claims.

Claims

1. A method for preparing a carboxyl fluorescent quantum dot-loaded gene editing system, characterized in that: The preparation method includes the preparation of functionalized carboxyl fluorescent quantum dots and the connection of functionalized carboxyl fluorescent quantum dots with plasmid DNA; wherein, The preparation of the functionalized carboxyl fluorescent quantum dots comprises: S1. Dispersing carboxyl fluorescent quantum dots in pure water, adding N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, and stirring the reaction at room temperature to activate the carboxyl groups on the surface of the carboxyl fluorescent quantum dots; S2. Add branched polyethyleneimine to the carboxyl-activated carboxyl fluorescent quantum dot reaction system and stir the reaction at room temperature; 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, and the obtained branched polyethyleneimine functionalized carboxyl fluorescent quantum dots, namely functionalized carboxyl fluorescent quantum dots, were stored at 4°C; The connection between the functionalized carboxyl fluorescent quantum dots and the plasmid DNA comprises: The plasmid DNA is diluted in deionized water, to which the functionalized carboxyl fluorescent quantum dots treated with ultrasound are added, and the mixture is incubated with shaking at room temperature to obtain a carboxyl fluorescent quantum dot-loaded gene editing system, which is subsequently used for gene editing of fertilized eggs of aquatic animals or cell transfection.

2. The preparation method according to claim 1, characterized in that: The preparation of the functionalized carboxyl fluorescent quantum dots comprises: S1. Disperse carboxyl fluorescent quantum dots with a carboxyl content of 6 μM / mL in pure water at 0.05 μM, add N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride at a final concentration of 12 μM / mL, and stir 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 the reaction at room temperature for 24 h; S3. The reaction system is dialyzed using a 7000D dialysis bag to remove unreacted N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and branched polyethyleneimine, and the obtained branched polyethyleneimine functionalized carboxyl fluorescent quantum dots are functionalized carboxyl fluorescent quantum dots, which are stored at 4° C. and will not aggregate within 90 days; The connection between the functionalized carboxyl fluorescent quantum dots and the plasmid DNA comprises: The plasmid DNA with a final concentration of 100 ng / μL was diluted in 100 μL of deionized water, and 100-400 μL of ultrasonically treated functionalized carboxyl fluorescent quantum dots were added thereto. The mixture was incubated at room temperature with shaking at 150 rpm for 30 min 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.

3. The preparation method according to claim 1 or 2, characterized in that: 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.

4. The preparation method according to claim 1 or 2, characterized in that: 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.

5. Carboxyl fluorescent quantum dot-loaded gene editing system, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 4.

6. Application of the carboxyl fluorescent quantum dot-loaded gene editing system according to claim 5 in gene editing of fertilized eggs of aquatic animals, characterized in that: The eggshells of the fertilized eggs are digested by an enzyme preparation.

7. The use according to claim 6, characterized in that: The aquatic animals include zebrafish, rare goby, grass carp, largemouth bass and coioidal grouper; the enzyme preparation is selected from one or more of trypsin, pronase, papain, keratinase and chymotrypsin.

8. The use according to claim 6, characterized in that: The carboxyl fluorescent quantum dot-loaded gene editing system is delivered to the fertilized eggs of aquatic animals by immersion, and the fertilized eggs of aquatic animals with the egg shells removed are immersed in the carboxyl fluorescent quantum dot-loaded gene editing system for 0.5-2 hours.

9. The use according to claim 8, characterized in that: The application concentration of the carboxyl fluorescent quantum dot-loaded gene editing system is 1-50 mg / L.

10. Use of the carboxyl fluorescent quantum dot-loaded gene editing system described in claim 5 in aquatic animal cell lines.

Images