A CRISPR / Cas9 delivery system and its applications

Abstract

This invention belongs to the field of genetic engineering technology and discloses a CRISPR / Cas9 delivery system and its applications. The invention uses fluorinated polyethyleneimine to load a CRISPR / Cas9 system, resulting in a CRISPR / Cas9 delivery system. Fluorinated polyethyleneimine can deliver the CRISPR / Cas9 vector into cells, exhibiting excellent transmembrane delivery efficiency and effectively mediating the entry of the CRISPR / Cas9 system into target cells. Applying this CRISPR / Cas9 delivery system to large-scale breeding of aquatic animals demonstrates high safety and excellent biocompatibility. This delivery system also exhibits superior gene editing efficiency and has broad application prospects in the field of gene editing breeding.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and relates to a CRISPR / Cas9 delivery system and application thereof. BACKGROUND

[0002] In recent years, the aquaculture industry in China has been expanding, and traditional breeding methods of aquatic animals relying on natural variation and artificial selection have the problems of long cycle and low directional improvement efficiency. It takes 10-15 years to breed a new variety with strong disease resistance and fast growth rate, and the improvement direction is limited to the natural gene pool, which is difficult to meet the demand of the industry for high-quality varieties. Gene editing technology can shorten the breeding cycle and significantly improve the directional improvement efficiency by precisely modifying specific gene sites, providing a solution to the problems faced by traditional breeding methods of aquatic animals.

[0003] However, only when the gene editing tool (such as CRISPR / Cas9 system) is accurately delivered to the target cells or embryos can the directional editing of the target gene be realized. Current gene editing of aquatic animals is mostly limited to microinjection technology, but microinjection requires a glass needle to penetrate the embryo membrane, which may cause physical damage and lead to abnormal development or death of the embryo. Moreover, microinjection relies on high-precision equipment and skilled operation, and the injection needs to be completed within a very short time of embryo development, resulting in a limited number of embryos that can be processed in a single experiment, which is difficult to meet the demand of the industry for large-scale breeding. Therefore, developing a gene editing delivery platform that takes into account both scalability and precision can solve the problems of embryo damage and low survival rate, low editing efficiency, and difficulty in large-scale breeding in aquatic animal breeding, accelerate the cultivation of high-quality aquatic animal new varieties, and promote the industrial application of gene editing technology in the aquaculture industry. SUMMARY

[0004] To solve the above technical problems, the present application provides a CRISPR / Cas9 delivery system and application thereof. The present application uses fluorinated polyethyleneimine to load the CRISPR / Cas9 system to obtain the CRISPR / Cas9 delivery system. Fluorinated polyethyleneimine can deliver the CRISPR / Cas9 vector into cells, has excellent transmembrane delivery efficiency, and can efficiently mediate the CRISPR / Cas9 system into target cells. The CRISPR / Cas9 delivery system of the present application is applied to large-scale breeding of aquatic animals, has high safety and excellent biocompatibility, has a better gene editing efficiency, and has a wide application prospect in the field of gene editing breeding.

[0005] In one aspect, the present application provides a CRISPR / Cas9 delivery system, which uses fluorinated polyethyleneimine to load the CRISPR / Cas9 system to obtain the CRISPR / Cas9 delivery system.

[0006] Furthermore, the preparation method of the fluorinated polyethyleneimine specifically includes the following steps:

[0007] S1: Polyethyleneimine (MW=1500) was dispersed in methanol at 2 mM, and 20 to 100 equivalents of 3-perfluorohexyl-1,2-epoxypropane were added. The mixture was stirred at room temperature for 48 h.

[0008] S2: The reaction product was dialyzed using a 1000 D dialysis bag to remove unreacted 3-perfluorohexyl-1,2-epoxypropane, yielding fluorinated polyethyleneimine.

[0009] Furthermore, fluorinated polyethyleneimine has a larger particle size than polyethyleneimine, resulting in a lower zeta potential.

[0010] Preferably, 60 equivalents of 3-perfluorohexyl-1,2-epoxypropane are added. Under these conditions, the nitrogen-to-fluorine ratio of the fluorinated polyethyleneimine prepared is 3.24:1.

[0011] Furthermore, the present invention uses fluorinated polyethyleneimine (F) with different degrees of fluorination modification. 20 ~F 100 FITC labeling was used, and the transmembrane efficiency of fluorinated polyethyleneimine in GCO cells was characterized by detecting fluorescence intensity. It was found that FITC... 60 The treated GCO cells exhibited the highest fluorescence intensity and good transmembrane efficiency, enabling efficient delivery of the CRISPR / Cas9 system into the cells.

[0012] Furthermore, the preparation method of the CRISPR / Cas9 delivery system includes the following steps:

[0013] Cas9 mRNA or Cas9 protein and sgRNA were dispersed in RNase-free H2O at a mass ratio of 3:1 to 5:1 to obtain a CRISPR / Cas9 system. Fluorinated polyethyleneimine was added to the system, and after thorough mixing, the mixture was incubated at room temperature for 5 to 15 minutes to obtain the CRISPR / Cas9 delivery system.

[0014] Specifically, the final concentration of fluorinated polyethyleneimine is 10~100 ng / μL, the final concentration of the CRISPR / Cas9 system is 10~100 ng / μL, and the mass ratio of fluorinated polyethyleneimine to the CRISPR / Cas9 system is 1:1.

[0015] On the other hand, the present invention seeks protection for the application of the above-mentioned CRISPR / Cas9 delivery system in large-scale breeding, including large-scale breeding of aquatic animals.

[0016] Further, the CRISPR / Cas9 delivery system is co-incubated with fertilized eggs of aquatic animals at room temperature for 0.5–4 hours to obtain gene-edited fertilized eggs. Alternatively, the CRISPR / Cas9 delivery system is co-incubated with sperm from aquatic animals, followed by artificial insemination to obtain gene-edited fertilized eggs, wherein the co-incubation time is not less than 0.5 hours; exemplaryly, the co-incubation conditions are 4°C for 1–4 hours or 28°C for 0.5–1 hour; the sperm is diluted by a factor of not less than 100.

[0017] Furthermore, the large-scale breeding includes large-scale gene editing of aquatic animals. atg5 Gene, tsg101 Gene.

[0018] Furthermore, this invention labels fluorinated polyethyleneimine with different degrees of fluorination modification using FITC, and characterizes the transmembrane efficiency of fluorinated polyethyleneimine in grass carp sperm by detecting fluorescence intensity. It was found that F... 60 The treated grass carp sperm showed a stronger fluorescence signal, indicating that F 60 Grass carp sperm exhibits good transmembrane efficiency.

[0019] Furthermore, this invention has shown through experiments that the Cas9 protein / sgRNA system loaded with fluorinated polyethyleneimine can successfully perform gene editing on grass carp fertilized eggs, with an indel efficiency of 76.4%. 60 PEI successfully delivered the Cas9 protein / sgRNA system into grass carp sperm, enabling large-scale gene editing of grass carp fertilized eggs via artificial insemination, with an indel efficiency of 64.7%. 60 The PEI-loaded Cas9 protein / sgRNA system successfully performed gene editing on zebrafish fertilized eggs, with an indel efficiency of 31.3%. 60 PEI successfully delivered the Cas9 protein / sgRNA system into zebrafish sperm, enabling large-scale gene editing of zebrafish fertilized eggs via artificial insemination, with an indel efficiency of 31.5%. 60 The PEI-loaded Cas9 protein / sgRNA system successfully performed gene editing on largemouth bass zygotes, with an indel efficiency of 23.3%. 60 PEI was able to successfully deliver the Cas9 protein / sgRNA system into the sperm of largemouth bass, and then achieve large-scale gene editing of largemouth bass fertilized eggs through artificial insemination, with an indel efficiency of 18.5%.

[0020] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0021] (1) This invention uses fluorinated polyethyleneimine to load the CRISPR / Cas9 system, obtaining a CRISPR / Cas9 delivery system. Fluorinated polyethyleneimine can deliver the CRISPR / Cas9 vector into cells, breaking through the delivery bottleneck of traditional vectors, possessing excellent transmembrane delivery efficiency, and can efficiently mediate the entry of the CRISPR / Cas9 system into target cells. This invention uses fluorinated polyethyleneimine (F...) with different degrees of fluorination modification... 20 ~F 100 FITC labeling was used, and the transmembrane efficiency of fluorinated polyethyleneimine in GCO cells was characterized by detecting fluorescence intensity. It was found that FITC... 60 The treated GCO cells exhibited the highest fluorescence intensity and good transmembrane efficiency, enabling efficient delivery of the CRISPR / Cas9 system into the cells. This invention labeled fluorinated polyethyleneimine with FITC at different degrees of fluorination modification and characterized the transmembrane efficiency of fluorinated polyethyleneimine in grass carp sperm by detecting fluorescence intensity. It was found that F... 60 The treated grass carp sperm showed a stronger fluorescence signal, indicating that F 60 The transmembrane efficiency is good in grass carp sperm. The fluorinated polyethyleneimine carrier of this invention has high safety, not only with excellent biocompatibility, but also effectively avoids the cytotoxicity and immunogenicity problems common in existing carriers. No obvious physiological damage was observed in zebrafish, grass carp and other models, laying a safe foundation for subsequent practical applications.

[0022] (2) The CRISPR / Cas9 delivery system of this invention exhibits superior editing efficiency when applied to large-scale breeding of aquatic animals. This delivery system not only demonstrates high application potential in zebrafish (model organism) and grass carp (economic species), but also provides a novel technical strategy for gene editing in other aquaculture species (such as shrimp, crab, and shellfish). It can directly serve the practical needs of aquatic animals in improving disease resistance, optimizing growth performance, and enhancing quality, and is of great significance for promoting the technological upgrading of the aquatic seed industry. Through experiments, this invention has found that F… 60 The loaded Cas9 protein / sgRNA system successfully performed gene editing on grass carp fertilized eggs, achieving an indel efficiency of 76.4%. 60 PEI successfully delivered the Cas9 protein / sgRNA system into grass carp sperm, and subsequently, through artificial insemination, enabled large-scale gene editing of grass carp fertilized eggs, achieving an indel efficiency of 64.7%. 60 The PEI-loaded Cas9 protein / sgRNA system successfully performed gene editing on zebrafish fertilized eggs, achieving an indel efficiency of 31.3%. 60PEI successfully delivered the Cas9 protein / sgRNA system into zebrafish sperm, enabling large-scale gene editing of zebrafish fertilized eggs via artificial insemination, with an indel efficiency of 31.5%. 60 The PEI-loaded Cas9 protein / sgRNA system successfully performed gene editing on largemouth bass fertilized eggs, achieving an indel efficiency of 23.3%. 60 PEI was able to successfully deliver the Cas9 protein / sgRNA system into the sperm of largemouth bass, and then achieve large-scale gene editing of largemouth bass fertilized eggs through artificial insemination, with an indel efficiency of 18.5%. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a photograph of fluorinated polyethyleneimine.

[0025] Figure 2 Figure showing the results of identifying the transmembrane efficiency of fluorinated polyethyleneimine with different degrees of fluorination modification in GCO cells.

[0026] Figure 3 Fluorinated polyethyleneimine F 60 Fourier transform infrared spectrum.

[0027] Figure 4 Fluorinated polyethyleneimine F 60 Comparison of particle size and zeta potential between polyethyleneimine and polyethyleneimine. Figure 4 In this context, A represents fluorinated polyethyleneimine F. 60 Comparison of particle size with polyethyleneimine; Figure 4 B in the text represents fluorinated polyethyleneimine F. 60 Comparison of Zeta potentials with those of polyethyleneimine.

[0028] Figure 5 For the FPEI-loaded Cas9 protein / sgRNA system for grass carp fertilized eggs atg5 A diagram showing the effect of gene editing.

[0029] Figure 6 Figure 1 shows the results of identifying the transmembrane efficiency of fluorinated polyethyleneimine with different degrees of fluorination modification in grass carp sperm.

[0030] Figure 7Fluorescence micrograph of grass carp sperm after treatment with PEI-loaded Cas9-EGFP protein / sgRNA-Cy3 system.

[0031] Figure 8 Treatment of grass carp sperm with FPEI-loaded Cas9 protein / sgRNA system for grass carp fertilized eggs atg5 A diagram showing the effect of gene editing.

[0032] Figure 9 For the FPEI-loaded Cas9 protein / sgRNA system for zebrafish fertilized eggs atg5 A diagram showing the effect of gene editing.

[0033] Figure 10 Treatment of zebrafish sperm with the FPEI-loaded Cas9 protein / sgRNA system for zebrafish fertilized eggs atg5 A diagram showing the effect of gene editing.

[0034] Figure 11 For the FPEI-loaded Cas9 protein / sgRNA system for testing largemouth bass fertilized eggs tsg101 A diagram showing the effect of gene editing.

[0035] Figure 12 Treatment of largemouth bass sperm with FPEI-loaded Cas9 protein / sgRNA system for the effects on largemouth bass fertilized eggs tsg101 A diagram showing the effect of gene editing. Detailed Implementation

[0036] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods and detection methods described in each embodiment are conventional methods; unless otherwise specified, the reagents and materials can be purchased commercially. Unless otherwise specified, the percentages in the following embodiments refer to mass percentages.

[0037] Polyethyleneimine was purchased from Gongbike New Material Technology (Shanghai) Co., Ltd., and 3-perfluorohexyl-1,2-epoxypropane was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0038] Example 1

[0039] This embodiment provides the synthesis of fluorinated polyethyleneimine (FPEI).

[0040] S1: Polyethyleneimine (PEI, MW=1500) was dispersed in methanol at a final concentration of 2 mM, and 20, 40, 60, 80, and 100 equivalents of 3-perfluorohexyl-1,2-epoxypropane were added respectively. The mixture was stirred at room temperature for 48 h.

[0041] S2: The reaction product was dialyzed using a 1000 D dialysis bag to remove unreacted 3-perfluorohexyl-1,2-epoxypropane. Dialysis was first performed with 50% methanol, followed by dialysis with pure water, ultimately yielding fluorinated polyethyleneimine (F) with different degrees of fluorination modification. 20 F 40 F 60 F 80 F 100 After passing through a 0.22 μm filter membrane, store at 4℃, as follows. Figure 1 As shown, fluorinated polyethyleneimine is a transparent solution in water and has good dispersibility.

[0042] Example 2

[0043] This embodiment provides a performance analysis of fluorinated polyethyleneimine.

[0044] 1. Determination of transmembrane efficiency

[0045] Fluorinated polyethyleneimine with different degrees of fluorination modification was labeled with Rhodamine B (Rhod B). After the reaction, Rhod B-labeled fluorinated polyethyleneimine was incubated with GCO cells under light-protected conditions at room temperature for 15 min. The fluorescence intensity in GCO cells after co-incubation with fluorinated polyethyleneimine with different degrees of fluorination modification was detected by flow cytometry. Figure 2 As shown, F 60 The treated GCO cells exhibited the highest fluorescence intensity and good transmembrane efficiency, enabling efficient delivery of the CRISPR / Cas9 system into the cells.

[0046] 2. Modification identification

[0047] Characterization of fluorinated polyethyleneimine F by Fourier transform infrared spectroscopy 60 The identification results are as follows Figure 3 As shown. By Figure 3 It can be seen that F 60 1350~1000 cm -1 The presence of a significant change in absorption peaks within the range indicates successful fluorination modification. Oxygen bomb combustion-ion chromatography analysis revealed a nitrogen content of 0.026% and a fluorine content of 0.01088%, with a nitrogen-to-fluorine ratio (molar ratio) of 3.24:1.

[0048] 3. Particle size and zeta potential

[0049] Analysis of fluorinated polyethyleneimine F using a Malvern nanolaser particle size analyzer 60 The particle size and zeta potential of polyethyleneimine were analyzed, and the changes in particle size and zeta potential of fluorinated polyethyleneimine were examined. The results are as follows: Figure 4 As shown. ByFigure 4 It can be seen that the particle size of fluorinated polyethyleneimine increases and the zeta potential decreases.

[0050] Example 3

[0051] This embodiment provides the application of a fluorinated polyethyleneimine-loaded CRISPR / Cas9 system in large-scale gene editing of grass carp fertilized eggs.

[0052] Cas9-EGFP protein (purchased from Beijing Qingke Biotechnology Co., Ltd.) and sgRNA-Cy3 (5'-GAGCCGTGTTTGAAGCGTGG-3') (synthesized by Beijing Qingke Biotechnology Co., Ltd.) were dispersed in RNase-free H2O at a mass ratio of 5:1 to obtain a CRISPR / Cas9 system with a final concentration of 10 ng / μL. Fluorinated polyethyleneimine of the same mass as the total mass of the CRISPR / Cas9 system was added to the system, with a final concentration of 20 ng / μL. After thorough mixing, the mixture was incubated at room temperature for 5-15 min to obtain the FPEI-loaded Cas9 protein / sgRNA system, which was ready for use within 30 min.

[0053] Grass carp zygotes were obtained through artificial insemination. After washing three times with RNase-free H2O, the washed zygotes were incubated at room temperature for 4 hours using an FPEI-loaded Cas9 protein / sgRNA system. The embryos were then placed in a recirculating aquaculture system for hatching. Simultaneously, the CRISPR / Cas9 system and fluorinated polyethyleneimine were delivered to the grass carp zygotes via microinjection. The concentration of Cas9-EGFP protein was 500 ng / μL, and the concentration of sgRNA-Cy3 was 100 ng / μL, with an injection volume of 5 nL. Embryo samples were collected after 24 hours. No obvious physiological damage was observed in the embryos. DNA was extracted using an alkaline lysis method, and the target gene was amplified by PCR. atg5 The primer sequences were F: 5'-TGGCAGATGACAAGGATGTGC-3', R: 5'-CGTTCTGCAGACCCATCCAC-3'. Sanger sequencing was used to analyze the efficiency of indels (insertions and deletions) generated by CRISPR editing, and the results are as follows: Figure 5 As shown. By Figure 5 It was found that the Cas9 protein / sgRNA system loaded with fluorinated polyethyleneimine could successfully edit the genes of grass carp fertilized eggs, with an indel efficiency of 76.4%. Embryos treated with microinjection showed no abnormalities, and gene editing of grass carp fertilized eggs was also successfully performed, indicating that FPEI does not affect the activity of the gene editing system.

[0054] Example 4

[0055] This embodiment provides the application of a fluorinated polyethyleneimine-loaded CRISPR / Cas9 system in processing grass carp sperm and subsequently scaling up gene editing of grass carp fertilized eggs.

[0056] 1. Identification of transmembrane efficiency of fluorinated polyethyleneimine with different degrees of fluorination modification in grass carp sperm

[0057] Fluorinated polyethyleneimine with different degrees of fluorination modification was labeled with FITC (fluorescein isothiocyanate). After the reaction, the FITC-labeled fluorinated polyethyleneimine was incubated with grass carp sperm under light-protected conditions at room temperature for 15 min. The fluorescence intensity of grass carp sperm after co-incubation with fluorinated polyethyleneimine with different degrees of fluorination modification was detected by flow cytometry. The transmembrane efficiency of fluorinated polyethyleneimine with different degrees of fluorination modification in grass carp sperm was analyzed. The results are as follows: Figure 6 As shown. By Figure 6 It can be seen that F 60 The treated grass carp sperm showed a stronger fluorescence signal, indicating that F 60 Grass carp sperm exhibits good transmembrane efficiency.

[0058] 2. Identification of the CRISPR / Cas9 delivery system in grass carp sperm using fluorinated polyethyleneimine

[0059] F was prepared according to the method in Example 4. 60 The PEI-loaded Cas9-EGFP protein / sgRNA-Cy3 system was used to incubate grass carp sperm on ice for 30 min. The effect of fluorinated polyethyleneimine on the delivery of the CRISPR / Cas9 system was observed by fluorescence microscopy. The results are as follows: Figure 7 As shown. By Figure 7 It can be seen that F 60 PEI successfully delivered Cas9-EGFP protein and sgRNA-Cy3 into grass carp sperm.

[0060] 3. Application of fluorinated polyethyleneimine-loaded CRISPR / Cas9 system in processing grass carp sperm and subsequently large-scale gene editing of grass carp fertilized eggs.

[0061] Mature male grass carp sperm were collected in RNase-free 1.5 mL centrifuge tubes and temporarily stored on ice in the dark. After activating the sperm with water, sperm motility was observed under a microscope. Once the sperm were confirmed to be healthy and motile, the next step of the experiment was performed. The grass carp sperm were diluted 200-fold with buffer solution (1 L: 10.0 g sodium citrate dihydrate, 0.3 g potassium chloride, 2.0 g sodium bicarbonate, 20 g glucose, RNase-free H2O, filtered through a 0.22 μm membrane and stored at 4°C). The sperm were then diluted with F... 60Grass carp sperm were incubated using a PEI-loaded Cas9 protein / sgRNA system at 4°C for 1 hour, followed by artificial insemination. The embryos were then placed in a recirculating aquaculture system for incubation. Embryo samples were collected after 24 hours. No obvious physiological damage was observed in the embryos. DNA was extracted using an alkaline lysis method, and the target gene was amplified by PCR. atg5 Sanger sequencing analysis was used to analyze the efficiency of indels (insertions and deletions) generated by CRISPR editing, and the results are as follows: Figure 8 As shown. By Figure 8 It can be seen that F 60 PEI was able to successfully deliver the Cas9 protein / sgRNA system into grass carp sperm, and then achieve large-scale gene editing of grass carp fertilized eggs through artificial insemination, with an indel efficiency of 64.7%.

[0062] Example 5

[0063] This embodiment provides the application of a fluorinated polyethyleneimine-loaded CRISPR / Cas9 system in large-scale gene editing of zebrafish zygotes.

[0064] F was prepared according to the method in Example 4. 60 PEI-loaded Cas9 protein / sgRNA system. Zebrafish zygotes were obtained through natural fertilization, washed three times with RNase-free H2O, and then subjected to F... 60 Fertilized eggs were incubated for 4 hours using a PEI-loaded Cas9 protein / sgRNA system, and then the embryos were placed in a recirculating aquaculture system for further incubation. Embryo samples were collected after 24 hours. No obvious physiological damage was observed in the embryos. DNA was extracted using an alkaline lysis method, and the target gene was amplified by PCR. atg5 Sanger sequencing analysis was used to analyze the efficiency of indels (insertions and deletions) generated by CRISPR editing, and the results are as follows: Figure 9 As shown. By Figure 9 It can be seen that F 60 The PEI-loaded Cas9 protein / sgRNA system successfully performed gene editing on zebrafish fertilized eggs, achieving an indel efficiency of 31.3%.

[0065] Example 6

[0066] This embodiment provides the application of a fluorinated polyethyleneimine-loaded CRISPR / Cas9 system in processing zebrafish sperm and subsequently scaling up gene editing of zebrafish zygotes.

[0067] F was prepared according to the method in Example 4. 60A PEI-loaded Cas9 protein / sgRNA system was used to collect mature male zebrafish sperm in RNase-free 1.5 mL centrifuge tubes, which were then temporarily stored on ice in the dark. After activating the sperm with water, sperm motility was observed under a microscope. Once sperm health and motility were confirmed, further experiments were performed. The zebrafish sperm were diluted 100-fold with buffer (same as in Example 5). Using F... 60 Zebrafish sperm were incubated using a PEI-loaded Cas9 protein / sgRNA system at 4°C for 1 hour, followed by artificial insemination. Embryos were then placed in a recirculating aquaculture system for incubation. Embryo samples were collected after 24 hours. No obvious physiological damage was observed in the embryos. DNA was extracted using an alkaline lysis method, and the target gene was amplified by PCR. atg5 Sanger sequencing analysis was used to analyze the efficiency of indels (insertions and deletions) generated by CRISPR editing, and the results are as follows: Figure 10 As shown. By Figure 10 It can be seen that F 60 PEI was able to successfully deliver the Cas9 protein / sgRNA system into zebrafish sperm, and then achieve large-scale gene editing of zebrafish fertilized eggs through artificial insemination, with an indel efficiency of 31.5%.

[0068] Example 7

[0069] This embodiment provides the application of a fluorinated polyethyleneimine-loaded CRISPR / Cas9 system in large-scale gene editing of largemouth bass zygotes.

[0070] F was prepared according to the method in Example 4. 60 PEI loaded with Cas9 protein / sgRNA system, pathogenic gene tsg101 The amplification primers for the gene were F: 5'-caaggacttgaagcctcttatgg-3', R: 5'-ccacagacacactgggatgttg-3'. The nucleotide sequence of the sgRNA was 5'-AGAGACCTGATGAGCTTGACAGG-3'. Largemouth bass zygots were obtained through natural fertilization, washed three times with RNase-free H2O, and then amplified using F... 60 After 4 hours, washed fertilized eggs were incubated using a PEI-loaded Cas9 protein / sgRNA system, and then the embryos were placed in a recirculating aquaculture system for further incubation. Embryo samples were collected after 24 hours. No obvious physiological damage was observed in the embryos. DNA was extracted using an alkaline lysis method, and the target gene was amplified by PCR. tsg101 Sanger sequencing analysis was used to analyze the efficiency of indels (insertions and deletions) generated by CRISPR editing, and the results are as follows: Figure 11 As shown. By Figure 11 It can be seen that F 60The PEI-loaded Cas9 protein / sgRNA system successfully performed gene editing on largemouth bass fertilized eggs, with an indel efficiency of 23.3%.

[0071] Example 8

[0072] This embodiment provides the application of a fluorinated polyethyleneimine-loaded CRISPR / Cas9 system in processing largemouth bass sperm and subsequently scaling up gene editing of largemouth bass fertilized eggs.

[0073] Preparation of F 60 A PEI-loaded Cas9 protein / sgRNA system was used to collect mature male largemouth bass sperm in RNase-free 1.5 mL centrifuge tubes, which were then temporarily stored on ice in the dark. After activating the sperm with water, sperm motility was observed under a microscope. Once sperm health and motility were confirmed, further experiments were performed. The largemouth bass sperm were diluted 100-fold with buffer (same as in Example 5). Using F... 60 Largemouth bass sperm were incubated in a PEI-loaded Cas9 protein / sgRNA system at 4°C for 1 h, followed by artificial insemination. Embryos were then placed in a recirculating aquaculture system for incubation. Embryo samples were collected after 24 h. No obvious physiological damage was observed in the embryos. DNA was extracted using an alkaline lysis method, and the target gene was amplified by PCR. tsg101 Sanger sequencing analysis was used to analyze the efficiency of indels (insertions and deletions) generated by CRISPR editing, and the results are as follows: Figure 12 As shown. By Figure 12 It can be seen that F 60 PEI was able to successfully deliver the Cas9 protein / sgRNA system into the sperm of largemouth bass, and then achieve large-scale gene editing of largemouth bass fertilized eggs through artificial insemination, with an indel efficiency of 18.5%.

[0074] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. Use of a CRISPR / Cas9 delivery system in mass selection breeding, characterized in that, The CRISPR / Cas9 delivery system is obtained by loading a CRISPR / Cas9 system with fluorinated polyethyleneimine; The fluorinated polyethyleneimine is obtained by fluorination modification of polyethyleneimine; The reagent used in the fluorination modification is 3-perfluorohexyl-1,2-epoxypropane; The molar ratio of 3-perfluorohexyl-1,2-epoxypropane to polyethyleneimine is 60:1; The CRISPR / Cas9 delivery system is obtained by adding fluorinated polyethyleneimine to a CRISPR / Cas9 system with a final concentration of 10 ng / μL, the amount of fluorinated polyethyleneimine being the same as the total mass of the CRISPR / Cas9 system, the final concentration of fluorinated polyethyleneimine being 20 ng / μL, fully mixing, and incubating at room temperature for 5-15 min. The large-scale breeding is large-scale breeding of aquatic animals, and the aquatic animals are grass carp.

2. Use according to claim 1, characterized in that, The CRISPR / Cas9 delivery system is incubated with the zygote of the aquatic animal at room temperature for 0.5-4 h to obtain a gene-edited zygote.

3. Use according to claim 1, characterized in that, The CRISPR / Cas9 delivery system is incubated with the sperm of the aquatic animal, and the gene-edited zygote is obtained by artificial insemination.

4. Use according to claim 3, characterized in that, The sperm is diluted before the incubation, and the dilution factor is not less than 100 times; The incubation time is not less than 0.5 h.

5. The use according to claim 1, characterized in that, The large-scale breeding includes large-scale genetic editing of aquatic animals atg5 genes, tsg101 genes.

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