A method, system, and composition for delivering macromolecules to the sperm of marine fish based on exosomes

Abstract

The present application relates to a method, system and composition for delivering macromolecules into the sperm of marine fish based on exosomes, belonging to the cross field of biotechnology and reproductive engineering, which utilizes exosomes displaying CD81 protein as a carrier, and through the specific binding of the CD81 protein with CD9 protein on the cell membrane of fish sperm, the biological macromolecules loaded in the exosomes are delivered into the sperm of marine fish. The present application is targeted and integrated with tracing: it can enhance the targeting by utilizing the potential interaction of CD81 and sperm surface protein CD9, and realize real-time tracing of the delivery process through EGFP fluorescence.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of biotechnology and reproductive engineering, and specifically relates to a method, system and composition for delivering macromolecules to sperm of marine fish based on exosomes. Background Technology

[0002] Exosomes are nanoscale vesicles secreted by cells, serving as natural intercellular communication carriers that protect and deliver bioactive substances such as proteins and nucleic acids to recipient cells. In the male reproductive system, the interaction between sperm and exosomes plays a crucial regulatory role in sperm maturation, capacitation, and fertilization capacity. However, how to utilize exosomes to achieve targeted and efficient delivery of exogenous functional macromolecules (such as gene editing components) into sperm remains a technological challenge. Existing sperm transfection methods, such as electroporation and liposome transfection, often cause significant damage to sperm motility and are inefficient. Summary of the Invention

[0003] The technical problem to be solved by this invention is to provide an efficient and controllable method for delivering biomolecules into fish sperm, especially macromolecules that are difficult to deliver using traditional methods such as gene editing systems.

[0004] This invention is achieved through the following technical solution: A method for delivering macromolecules to marine fish sperm via exosomes utilizes exosomes displaying CD81 protein as carriers. The method involves the specific binding of the CD81 protein to a ligand-receptor of the CD9 protein on the sperm cell membrane of the fish, thereby delivering the biomolecules loaded within the exosomes into the sperm of the marine fish. The amino acid sequence of the CD81 protein is shown in SEQ ID NO.5, and the amino acid sequence of the CD9 protein is shown in SEQ ID NO.6.

[0005] Furthermore, the exosomes displaying the CD81 protein are obtained by introducing the CD81 gene into mammalian packaging cells, culturing them, and then separating and extracting them from their culture supernatant.

[0006] Furthermore, the biomolecule is a CRISPR / Cas ribonucleoprotein complex.

[0007] Furthermore, electroporation was used to load the biomacromolecule, a CRISPR / Cas ribonucleoprotein complex, into exosomes displaying the CD81 protein.

[0008] A system for delivering biomacromolecules to fish sperm, the system comprising: Vector component: It is a delivery vector composed of exosomes displaying CD81 protein; Targeting component: It is a receptor composed of CD9 protein inherent in the sperm cell membrane of fish; The delivery vector achieves targeted delivery of the biomolecules through the specific binding of CD81 and CD9.

[0009] Furthermore, the exosomes are derived from 293T cells.

[0010] Furthermore, the CD81 is fused with a reporter gene (such as EGFP).

[0011] The present invention also provides a composition for delivering macromolecules to marine fish sperm based on exosomes, the composition comprising exosomes loaded with biomolecules and displaying CD81 protein, the biomolecules being selected from proteins and / or CRISPR / Cas ribonucleoprotein complexes.

[0012] Furthermore, the CD81 is fused with a reporter gene (such as EGFP).

[0013] The present invention also provides the application of the method or system in fish gene editing breeding.

[0014] The present invention also provides a method for screening or evaluating pairing proteins that can mediate the delivery of biomolecules from exosomes to fish sperm, wherein the pairing proteins are CD81 and its interacting protein CD9.

[0015] The beneficial effects of the present invention compared with the prior art are: (1) The present invention integrates targeting and tracing: the CD81-EGFP fusion protein displayed on the exosome membrane can enhance targeting by utilizing the potential interaction between CD81 and sperm surface protein CD9, and can also achieve real-time tracing of the delivery process through EGFP fluorescence.

[0016] (2) High-efficiency loading: The present invention uses electroporation physical method to overcome the bottleneck of low efficiency of spontaneous loading of macromolecules by exosomes, and is especially suitable for loading macromolecular complexes such as CRISPR / Cas9 RNP.

[0017] (3) Sperm-friendly: The exosomes of this invention are natural biological carriers, and their incubation with sperm has a much smaller impact on sperm motility than traditional physical and chemical transfection methods, thus preserving the possibility for subsequent fertilization and other applications.

[0018] (4) Wide range of applications: The method of this invention is not limited to the delivery of gene editing systems. In principle, it can deliver any protein, RNA and other molecules that can be loaded into exosomes through electroporation. The platform has strong scalability. Attached Figure Description

[0019] Figure 1 Image of 293T cells transfected with CD81-N1 plasmid; Figure 2 Expression of EGFP protein in CD81-Exos; Experimental group: CD81-N1-Exos; Negative control: N1-Exos; Positive control: 293-N1-cell; Figure 3 Diagram showing the binding of CD9 antibody to sperm; Figure 4 A diagram showing the fusion of sperm with CD81-N1-Exos after CD9 antibody blockade; Figure 5 Expression diagram of CD81 and EGFP proteins in sperm after CD9 antibody blockade; Figure 6 Figure A shows the efficiency of RNP loading on exosomes; A is a Western blotting plot of Cas9 protein standards and CD81-Exos-RNP samples at different concentrations, and B is a standard curve of Cas9 protein. Figure 7 A diagram validating the delivery of CD81-Exos-RNP to sperm. Detailed Implementation

[0020] The present invention will now be described in conjunction with specific embodiments. In this embodiment, marine fish are used as the research object, and all experimental supplies involved were purchased from the market unless otherwise specified.

[0021] Example 1: Exosome delivery of CD81-EGFP protein to fish sperm This embodiment aims to establish and validate a basic method for delivering functional proteins (such as EGFP reporter protein) into fish sperm cells via exosomes displaying the CD81 protein (CD81-Exos), and to demonstrate that the delivery mechanism depends on the specific interaction between the exosomal membrane protein CD81 and the sperm cell membrane protein CD9.

[0022] 1. Materials and Methods 1.1 Plasmid Construction Primer design and synthesis: Specific primer pairs were designed and synthesized with the sequences SEQ ID NO.1 (5'-CCGGAGCTCATGGGAGTCGAAGGCTGCACCAA-3') and SEQ ID NO.2 (5'-CCGGTCGACTGGTACACTGGGCTGTTGCGGA-3'), which contain ScaⅠ and SalⅠ restriction sites.

[0023] Gene amplification: Using cDNA extracted from the ovarian tissue of marine fish (e.g., large yellow croaker) as a template, PCR amplification was performed using the above primer pairs to obtain the coding region fragment of the CD81 gene.

[0024] Vector Construction: The PCR amplification product and the eukaryotic expression vector pEGFP-N1 were double-digested with ScaI and SalI, respectively, followed by ligation using T4 DNA ligase. The ligation product was transformed into *E. coli* DH5α competent cells and screened on LB agar plates containing kanamycin. Single colonies were picked and amplified by shaking. The plasmid was extracted and its sequence was verified by DNA sequencing. The correct recombinant plasmid was obtained and named CD81-N1 (its pattern can be found in [reference needed]). Figure 1 ).

[0025] 1.2 Preparation and Identification of Exosomes Cell transfection and culture: Human embryonic kidney cells (293T cells) in the logarithmic growth phase were seeded in culture dishes, and the CD81-N1 plasmid was transfected into the cells using liposome transfection. After transfection, the cells were cultured in a dedicated medium without exosome serum for 24 hours.

[0026] Exosome extraction: Collect the cell culture supernatant and perform the following centrifugation steps sequentially: centrifuge at 500 ×g for 10 minutes to remove cells; centrifuge at 2,000 ×g for 10 minutes to remove dead cells; centrifuge at 10,000 ×g for 30 minutes to remove cell debris; finally, ultracentrifuge the supernatant at 140,000 ×g for 90 minutes at 4°C. The precipitate is the crude exosome extract. Resuspend the precipitate in pre-cooled PBS to obtain the CD81-Exos suspension.

[0027] Exosome identification: Extracted exosomes were identified using Western blotting. Results are as follows: Figure 2 As shown, analysis of the experimental group CD81-N1-Exos, the negative control N1-Exos, and the positive control 293-N1-cell demonstrated that the CD81-N1-Exos group contained EGFP protein, and exosomes displaying the CD81-EGFP fusion protein were successfully obtained.

[0028] 1.3 Verification of CD9 protein on sperm surface Fresh marine fish sperm (sperm from Sebastes schlegelii used in this and the following examples) were incubated with anti-CD9 antibody (1:1000) and FITC-labeled secondary antibody. Under a confocal microscope, clear green fluorescence was observed on the surface of the sperm. Figure 3 This demonstrates the presence of the CD81 interacting protein on the sperm surface, providing a possible site for exosome binding.

[0029] 1.4 Verification of exosomal protein delivery efficiency Experimental group (CD9 blocking group): Fresh sperm were first incubated with CD9 antibody (1:1000) for 2 hours to block CD9 protein on the sperm surface. Then the sperm were washed 3 times with PBST and then co-incubated with CD81-Exos suspension for 2 hours.

[0030] Control group: Fresh sperm were directly incubated with an equal volume of CD81-Exos suspension for 2 hours.

[0031] 2. Detection and Results Laser confocal microscopy observation: After incubation, both groups of sperm were thoroughly washed with PBS (3 times) to remove surface-attached exosomes. Observation revealed a clear EGFP green fluorescent signal inside the control group sperm. Figure 4 ( ), while the fluorescence signal of sperm in the experimental group was significantly weakened.

[0032] Western Blot Validation: Total protein was extracted from both groups of sperm using RIPA lysis buffer. After quantification using the BCA method, 10 μg of protein was taken for Western Blot analysis. Results are as follows: Figure 5 As shown, specific bands of CD81 and EGFP were detected in the sperm lysate of the control group, while the band signals in the experimental group were significantly weakened.

[0033] The above results demonstrate that CD81-Exos can effectively deliver CD81 protein into sperm, and this process depends in part on the CD9 protein on the sperm surface.

[0034] Example 2: Exosome electroporation loading of CRISPR / Cas9 system and its delivery to fish sperm This embodiment details the core gene editing system delivery method of the present invention, namely, loading the CRISPR / Cas9 ribonucleoprotein complex into CD81-Exos via electroporation, and verifying its effectiveness in delivering it into fish sperm.

[0035] 1. Preparation of RNP complex Single-stranded guide RNAs were designed and chemically synthesized targeting specific genes (such as the tyr gene) in marine fish. The tyr sgRNA sequence is: P1 (SEQ ID NO.3): taatacgac tcactataGGTGGGGGACCGGAGTCCACgttttagagctagaa. P2 (SEQ ID NO. 4): AGCACCGACTCGGTGCCACT.

[0036] The purified Cas9 protein was incubated with sgRNA at a molar ratio of 1:1 at room temperature for 5 minutes to form an RNP complex with DNA cleavage activity.

[0037] 2. Extraction of exosomes The method was the same as in Section 1.2 of Example 1, in which CD81-Exos was extracted from the culture supernatant of 293T cells transfected with CD81-N1 plasmid and resuspended in sterile PBS.

[0038] 3. Electroporation method for loading RNP Take 50 μg of CD81-Exos suspension and gently mix it with all the RNP complexes prepared above, then transfer the mixture to a pre-cooled 0.4 cm electroporation cuvette. Use an electroporator with the following parameters: voltage 120 V, pulse width 0.1 ms, 20 consecutive pulses. Immediately after electroporation, incubate the sample at 37°C for 30 minutes to allow the exosome membrane to repair. Centrifuge the recovered mixture at 4°C and 140,000 ×g for 90 minutes, discard the supernatant to remove unloaded free RNPs, and resuspend the precipitate in 50 μL of PBS to obtain exosomes loaded with the CRISPR / Cas9 system (CD81-Exos-RNP), which are stored at 4°C for later use.

[0039] 4. Quantitative determination of loading efficiency Western Blot Detection: Standards (100 ng, 200 ng, 400 ng, 800 ng) were prepared from purified Cas9 protein. 10 μL of CD81-Exos-RNP sample was taken together with the standards and subjected to Western Blot analysis using an anti-Cas9 antibody.

[0040] Gray-scale analysis and calculation: After scanning the Western blot results, the gray-scale values ​​of each band were measured using ImageJ image analysis software. A standard curve was plotted with the concentration of Cas9 protein standards on the x-axis and the gray-scale values ​​on the y-axis. Figure 6 Substituting the gray values ​​of the CD81-Exos-RNP bands into the standard curve equation, the absolute amount of Cas9 protein loaded in the exosomes was calculated.

[0041] Loading efficiency calculation: Loading efficiency (%) = (Amount of Cas9 protein loaded in exosomes / Total amount of Cas9 protein initially added) × 100%. Based on three independent replicate experiments, the average loading efficiency of the RNP complex into exosomes was 51.5%. Figure 6 This demonstrates the effectiveness of the electroporation loading method.

[0042] 5. Validation of CD81-Exos-RNP delivery to sperm Sperm treatment and incubation: Fresh marine fish sperm were collected and washed twice with PBS.

[0043] Experimental group: Sperm and 50 μL CD81-Exos-RNP were incubated in 500 μL PBS at room temperature in the dark for 2 hours, with gentle shaking during the incubation period.

[0044] Control group: Sperm were incubated with an equal amount of “empty” CD81-Exos without RNP loading under the same conditions.

[0045] Washing and fixation: After incubation, the sperm were washed three times with PBS containing 0.1% BSA (centrifuged at 500 × g for 5 minutes each time) to thoroughly remove exosomes that had not entered the sperm. Then, the sperm were fixed with 4% paraformaldehyde for 10 minutes.

[0046] Observations and Results: After fixation, sperm cells were washed with PBS, mounted with DAPI mounting medium, and observed under a laser confocal microscope. Figure 7 As shown, the sperm in the experimental group exhibited a clear fluorescent signal, while the sperm in the control group only showed blue DAPI fluorescence. This result directly confirms that the CD81-Exos-RNP prepared in this invention can efficiently deliver a bioactive CRISPR / Cas9 gene editing system into fish sperm.

[0047] Amino acid sequence of CD81 (SEQ ID NO.5): MGVEGCTKCIKYLLFFFNFIFWLAGGVILGVA LWLRHDPKTSNLLELEFDGNPAPSTFYISVHILIAVGAVMMVVGFLGCYGAIQESQCLLGTFACLVILFA CEVAAGIWGFMHKDTVSGEMINYYNTVYEKAMVESVTE QDKTKAAASVLKVFHETLSCCGKGKGTSV LTRLTDTLGLTDICPSSGATTDCHSRITELFTDKIYLIGIAALVVAVIMIFEMIFSMVLCCGIRNSPVY.

[0048] Amino acid sequence of CD9 (SEQ ID NO.6): MTALSSWELCVKYALFIFNFVFW LAGTAVLAVGLWLRFDSRTAALFEGEDSPTVFFTGVYILVAAGALLMMVVGFLGCCGAIKESPCMLGLFF FFLLVIFAAEVAAGIWGLSNKDRVVEEVTEFYKQTYTNYKDTKQEALKETLRLIHFGLDCCGPTGTVIDAAKDICPKKEGLEVLITTSCPAAIDEVFNNKLHIIGGVGIGIGVIMIFGMIFSMMLCCAIKKSRDFV.

Claims

1. A method for delivering macromolecules to sea water fish spermatozoa based on exosomes, characterized in that, The application discloses a method for delivering biological macromolecules into the interior of marine fish sperm by using exosomes displaying CD81 protein as a carrier, and through ligand-specific binding of the CD81 protein to CD9 protein on the membrane of fish sperm cells, wherein the amino acid sequence of the CD81 protein is shown as SEQ ID NO. 5, the amino acid sequence of the CD9 protein is shown as SEQ ID NO. 6, and the marine fish sperm is Sebastes hubbsi sperm. The exosomes displaying the CD81 protein are obtained by introducing a CD81 gene into a mammalian packaging cell, and then separating and extracting the exosomes from the culture supernatant of the mammalian packaging cell after culture. The biological macromolecule CRISPR / Cas ribonucleoprotein complex is loaded into the exosomes displaying the CD81 protein by using an electroporation method.

2. Application of the method in claim 1 in fish gene editing breeding, and the fish is Sebastes hubbsi.

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