An electroporation-mediated fish skin tissue electroporation or gene editing method

Abstract

The present application relates to a kind of electroporation-mediated fish skin tissue electric transformation or gene editing method, belong to gene editing technical field.The technical problem solved by the present application is to provide electroporation-mediated fish skin tissue electric transformation or gene editing method.The method is after fish body is anaesthetized, and external molecule is dropped to fish body surface, and micro-needle roller is used to carry out transverse rolling and longitudinal rolling in turn on fish body surface;Subsequently, external molecule is delivered to fish skin tissue by electroporation.The present application realizes the efficient delivery of external molecule by the combination method of " micro-needle roller pretreatment + optimization of electroporation parameter", overcomes the limitation in the delivery of fish skin tissue in prior art, provides effective technical means for further studying the function of skin pigment cell, analyzing the molecular mechanism of body color regulation, and fish economic character optimization, with wide application prospect.

Description

Technical Field

[0001] This invention relates to an electroporation-mediated electrotransfection or gene editing method for fish skin tissue, belonging to the field of gene editing technology. Background Technology

[0002] The market value and quality of the leopard-gill perch (Plectropomus leopardus) are largely influenced by its body coloration. Body color is not only an important biological characteristic for fish in terms of camouflage, protection, and thermoregulation, but also a key appearance indicator for consumers to judge its quality and determine its market price. The formation of fish body color is a complex biological process, essentially dependent on the types, numbers, and distribution of pigment cells in the skin tissue, as well as the migration and deposition of pigment granules within the cells. Therefore, in-depth analysis of the regulatory mechanisms of body color formation at the molecular level, and based on this, precise intervention of key genes using modern biotechnology, has become a core research direction for enhancing the economic value of the leopard-gill perch and achieving genetic trait improvement.

[0003] With the rapid development of molecular biology techniques, CRISPR / Cas9 gene editing technology, as well as gene overexpression or RNA interference based on exogenous plasmid DNA, have become powerful tools for studying gene function and conducting genetic modification. In fish research, conventional methods for delivering these exogenous macromolecules (such as Cas9 / gRNA ribonucleoprotein complexes or plasmid DNA) mainly include liposome transfection, nanoparticle encapsulation delivery, and microinjection. However, these methods have significant limitations when applied to fish skin tissue delivery: the delivery efficiency of liposomes and nanoparticles in skin tissue is generally low, and there are currently no reports of their application in fish skin tissue; while microinjection technology is generally only suitable for early embryos, which are difficult to achieve in marine fish embryos, including the leopard gill sea bass, due to their hard eggshells and osmotic pressure. Therefore, existing technologies cannot effectively support efficient gene function research and genetic manipulation in fish skin tissue.

[0004] Electroporation is a physical-based macromolecule delivery method that uses high-voltage electrical pulses to create transient hydrophilic micropores in the cell membrane, allowing exogenous nucleic acids and other macromolecules to pass through the membrane structure and enter the cell. This technique has proven effective in delivering macromolecules to various fish tissues, including muscle and retina, and is commonly used to mediate the introduction of plasmid DNA or ribonucleoprotein complexes (RNPs) for heterologous expression or editing of target genes. The success of these applications largely stems from the structural characteristics of these tissues—relatively loose cell arrangement and a lack of a keratinized outer layer—allowing electrical pulses to act more easily on the cell membrane, thus exhibiting high delivery efficiency.

[0005] However, fish skin tissue possesses unique physiological characteristics. As the body's first line of defense against the external environment, the skin's outermost layer is covered by a highly keratinized cell layer, beneath which lie tightly packed layers of epidermal cells, rich in mucus cells and scales, forming a multi-layered physiological barrier. This structure not only possesses strong physical barrier capabilities but also forms a chemical defense through mucus secretion, greatly limiting the penetration and absorption of exogenous macromolecules.

[0006] If electroporation conditions applicable to tissues other than skin (such as muscle tissue) are directly transferred to skin tissue, the following problems arise:

[0007] Different structural barriers: The tight junctions between the stratum corneum and epidermal cells significantly reduce the effectiveness of electrical pulses.

[0008] Parameter mismatch: The voltage and duration of electrical pulses commonly used in other tissues are insufficient for effective molecular penetration in skin tissue, and even increasing the voltage can easily cause irreversible damage.

[0009] Experimental evidence: Without skin pretreatment, the inventors directly dropped the plasmid onto the skin surface and applied an electric field, but failed to detect an effective delivery signal.

[0010] The above factors together make it difficult to apply traditional electroporation strategies to fish skin tissue, resulting in a near-complete lack of application of this technology in gene function research and genetic manipulation, thus limiting the development of related fields.

[0011] Therefore, developing an efficient and safe exogenous macromolecule delivery technology targeting the unique barrier structure of fish skin has become an urgent need in this field. An ideal solution should overcome the limitations of existing delivery methods, providing a reliable tool for the efficient transfer of gene editing elements and expression vectors into skin tissue, thereby promoting basic research on the mechanisms of fish body color formation and the precise genetic improvement of economically important traits. Summary of the Invention

[0012] To address the above deficiencies, the technical problem solved by this invention is to provide an electroporation-mediated electrotransfection or gene editing method for fish skin tissue, enabling efficient and safe delivery of exogenous macromolecules (such as gene editing components or expression plasmids).

[0013] The present invention relates to an electroporation-mediated method for electrotransfection or gene editing of fish skin tissue, comprising the following steps:

[0014] After anesthetizing the fish, exogenous molecules were dropped onto the fish surface, and a microneedle roller was used to roll the molecules laterally and longitudinally on the fish surface. Electroporation was then performed to deliver the exogenous molecules into the fish's skin tissue. The electroporation parameters were as follows: puncture pulse: voltage 30–60 V, length 9–11 ms, interval 95–105 ms, 2–4 pulses, attenuation rate 8–12%, positive polarity; transfer pulse: voltage 18–22 V, length 70–80 ms, interval 45–55 ms, 4–6 pulses, attenuation rate 35–45%, alternating positive / negative polarity; the number of electric shocks was 1–3.

[0015] This invention, targeting the unique structure of skin tissue, particularly that of the leopard-gill spiny perch, employs a combination of microneedle roller pretreatment and optimized electroporation parameters. By optimizing delivery conditions for electroporation, including pulse voltage, pulse duration, pulse interval, and pulse count, a gene editing method suitable for skin tissue is constructed. This method utilizes brief electrical pulses to enhance cell membrane permeability, achieving efficient delivery of exogenous molecules (plasmids and Cas9 / gRNA complexes), overcoming the limitations of existing technologies in fish skin tissue delivery. This technology provides an effective technical means for further research on the function of skin pigment cells, elucidating the molecular mechanisms of body color regulation, and optimizing economic traits in fish, and has broad application prospects.

[0016] In one specific embodiment of the present invention, the fish is a leopard-gill spiny perch.

[0017] The exogenous molecule can be a gene editing component or expression plasmid commonly used in the art. In one specific embodiment of the present invention, the exogenous molecule is an EGFP plasmid or a Cas9 / gRNA complex targeting the tyr gene.

[0018] In one embodiment of the present invention, the exogenous molecule for gene editing is a Cas9 / gRNA complex targeting the tyr gene. Delivering the Cas9 / gRNA complex targeting the tyr gene to skin tissue containing melanocytes via electroporation activates the CRISPR / Cas9 system, enabling precise editing of the key melanocyte gene tyr.

[0019] In one specific embodiment of the present invention, the length of the microneedle of the roller is 0.4 to 0.6 mm, the number of transverse rolling cycles is 8 to 12, and the number of longitudinal rolling cycles is 8 to 12.

[0020] In one embodiment of the present invention, the length of the microneedle of the roller is 0.5 mm, the number of transverse rolling is 10 times, and the number of longitudinal rolling is 10 times.

[0021] In one embodiment of the present invention, the parameters of electroporation are as follows: piercing pulse: voltage 40 V, length 10 ms, interval 100 ms, 3 pulses, attenuation rate 10%, positive polarity; transfer pulse: voltage 20 V, length 75 ms, interval 50 ms, 5 pulses, attenuation rate 40%, alternating positive / negative polarity; the number of electric shocks is 2.

[0022] This invention provides an electroporation-mediated electrotransfection or gene editing method for fish skin tissue, which can be used for the precise editing of the target gene *tyr* in *Leopard leopard perch*. Specifically, the method for precise editing of the *tyr* target gene in *Leopard leopard perch* involves delivering a Cas9 / gRNA complex targeting the *tyr* gene into the skin tissue of adult *Leopard leopard perch* using the aforementioned electroporation-mediated electrotransfection or gene editing method, thereby achieving precise editing of the *tyr* gene.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The method of this invention, through the combination of "microneedle roller pretreatment + optimized electroporation parameters", overcomes the bottleneck of traditional delivery methods that are difficult to penetrate the skin barrier, and achieves efficient delivery of exogenous molecules in fish skin tissue. While ensuring delivery efficiency, it significantly reduces irreversible damage, providing a new technical path for the precise editing and gene regulation of key genes in fish skin tissue.

[0025] The method of this invention can successfully deliver the Cas9 / gRNA complex to the skin tissue containing melanocytes via electroporation and achieve precise editing of the target gene tyr, enabling in-depth research on the molecular mechanisms of pigment cell function and body color formation; at the same time, it utilizes exogenous plasmid DNA to achieve overexpression or RNA interference of the target gene, elucidating its regulatory role on pigment cell function.

[0026] This invention marks the first time gene editing has been achieved in fish skin tissue, pioneering a new approach for conducting gene editing research directly in target tissues of live fish. This method not only provides an innovative tool for optimizing body color, verifying gene function, and improving the breed of the leopard-gill spiny perch, but also offers valuable insights for molecular biology research and the application of gene editing technology in the skin tissues of other fish species, demonstrating broad application prospects. Attached Figure Description

[0027] Figure 1 Figures show EGFP plasmid expression and bright-field morphology in the skin tissue of the leopard-gill spiny perch. The left column (A, C, E) shows fluorescence micrographs, and the right column (B, D, F) shows the corresponding bright-field images. A and B represent the experimental group; C and D represent negative control group 1; E and F represent negative control group 2. The scale bars for figures A, B, C, D, E, and F are all 250 μm.

[0028] Figure 2 Figures show the expression of EGFP plasmid and its bright-field morphology in the skin tissue of *Scourgeaurus leopardus*, Comparative Example 1 and Comparative Example 2. The left column (A, C) shows fluorescence micrographs, and the right column (B, D) shows the corresponding bright-field images. A and B represent Comparative Example 1, and C and D represent Comparative Example 2. The scale bars for Figures A, B, C, and D are all 2 mm.

[0029] Figure 3 The image shows the bright-field morphology of melanocytes in the skin tissue of the leopard-gill spiny perch. A and B are the experimental groups, and C and D are the negative control groups. A and C are on day 1 after electroporation, and B and D are on day 5 after electroporation. E is a magnified view of part of Figure B. The scale bar for Figures A, B, C, D, and E is 250 μm.

[0030] Figure 4 Image showing the T7E1 restriction enzyme digestion identification results of tyr-edited skin tissue from the leopard gill spiny perch. Detailed Implementation

[0031] The specific embodiments of the present invention will be further described below with reference to examples, but the present invention is not limited to the scope of the embodiments described herein.

[0032] Example 1

[0033] The following method was used to deliver the EGFP plasmid into the skin tissue of the leopard-gill spiny perch:

[0034] (1) Fish selection: Select 4-month-old leopard gill spiny perch.

[0035] (2) Preparation before the experiment:

[0036] EGFP plasmid extraction: E. coli containing the EGFP plasmid were cultured overnight in a medium containing appropriate antibiotics (12-16 h at 37°C and 200-250 rpm on a shaker) to ensure abundant bacterial growth. When an appropriate bacterial density was reached (i.e., OD600 ≈ 2-3), the bacteria were collected from the medium by centrifugation. The plasmid was extracted using a plasmid medium-volume extraction kit (general type) (plasmid concentration at least 1000 ng / μL); ready for use.

[0037] (3) Electroporation Experiment Procedure: After anesthetizing the leopard-gill spiny perch with MS-222, the EGFP plasmid to be delivered was dropped onto the surface of the fish using a precision syringe (hamilton, USA). A roller with a microneedle length of 0.5 mm was used to perform 10 transverse rolling and 10 longitudinal rolling on the surface of the fish. Electroporation was then performed using a NEPA21 super electroporator (Nepa Gene, China, Japan) and a CUY650P3 electrode (Nepa Gene). The following parameters were set for electroporation: PoringPulse: voltage 40 V, length 10 ms, interval 100 ms, 3 pulses, attenuation rate 10%, positive polarity; TransferPulse: voltage 20 V, length 75 ms, interval 50 ms. The experiment involved 5 pulses per second (ms), with a 40% attenuation rate and alternating positive and negative polarities. Two electroporations were performed. A negative control group 1 (pretreated with a microneedle roller but without electroporation) and a negative control group 2 (electroporated but without microneedle roller pretreatment) were also included. On day 1 post-experiment, the fluorescence signal in the EGFP delivery region was observed under a microscope, and the experimental group and the negative control group were compared and analyzed. The results are shown below. Figure 1 .

[0038] Figure 1 Figures show EGFP plasmid expression and bright-field morphology in the skin tissue of the leopard-gill spiny perch. The left column (A, C, E) shows fluorescence micrographs, and the right column (B, D, F) shows the corresponding bright-field images. Experimental groups A and B: EGFP plasmid delivered via electroporation after microneedle roller pretreatment; arrows indicate EGFP-positive cells. Negative control groups 1 C and D: Pretreated with microneedle roller but without electroporation; Negative control groups 2 E and F: Performed electroporation but without microneedle roller pretreatment. The scale bars for Figures A, B, C, D, E, and F are 250 μm.

[0039] The results showed that a significant enhancement of EGFP fluorescence signal in the target area could be observed on the first day of electroporation, with more efficient and uniform expression than the negative control group that was not treated with electroporation, indicating that the method of the present invention successfully delivered EGFP plasmid in skin tissue.

[0040] Comparative Examples: To further illustrate the criticality and superiority of the parameter range claimed in this invention, several comparative experiments were conducted. The following comparative examples use electroporation parameters and methods in zebrafish retina and muscle tissue to demonstrate that the efficient delivery described in this invention cannot be obtained outside the parameter range defined in the claims.

[0041] Comparative Example 1 (Low Voltage Group)

[0042] The remaining operations are the same as in Example 1, except for the electroporation parameters used. The electroporation parameters used in this comparative example are: piercing pulse: voltage 30 V, length 30 ms, interval 450 ms, 3 pulses, attenuation rate 10%, positive polarity; transfer pulse: voltage 10 V, length 50 ms, interval 450 ms, 5 pulses, attenuation rate 40%, positive polarity; the number of electric shocks is 1. The results are shown in […]. Figure 2 .

[0043] Figure 2 Figures 1 and 2 show EGFP plasmid expression and bright-field morphology in the skin tissue of the leopard-gill spiny perch. The left column (A, C) shows fluorescence micrographs, and the right column (B, D) shows the corresponding bright-field images. A and B are comparative examples 1, and C and D are comparative examples 2. The scale bars for figures A, B, C, and D are all 2 mm.

[0044] Experimental results showed that the EGFP fluorescence signal was almost invisible, and the proportion of EGFP-positive cells was low; no obvious irreversible damage was observed in the skin tissue. This indicates that excessively low voltage cannot achieve effective delivery.

[0045] Comparative Example 2 (High Voltage Group)

[0046] The remaining operations are the same as in Example 1, except for the electroporation parameters used. The electroporation parameters used in this comparative example are: piercing pulse: voltage 100 V, length 20 ms, interval 50 ms, 3 pulses, attenuation rate 10%, positive polarity; transfer pulse: voltage 50 V, length 70 ms, interval 50 ms, 5 pulses, attenuation rate 40%, alternating positive / negative polarity; the number of shocks is 2. The results are shown in […]. Figure 2 .

[0047] Experimental results showed that the proportion of EGFP-positive cells under high-voltage treatment was higher than that under low-voltage treatment, but still significantly lower than the proportion of EGFP-positive cells obtained in the embodiments of this invention. Further observation revealed that the tissue under this condition showed obvious and irreversible damage and necrosis. This indicates that high voltage may temporarily promote plasmid entry, but excessive electric field strength leads to large-scale cell death or loss of function, thereby significantly reducing the final detectable proportion of positive cells.

[0048] In summary, the experimental results show that outside the electroporation parameter range and roller assistance defined in this invention, the experimental results are either insufficiently efficient or excessively damaging, failing to balance high efficiency and minimal damage. These results further highlight the rationality and inventiveness of this invention, enabling the safe and efficient delivery of exogenous macromolecules to fish skin tissue.

[0049] Example 2

[0050] The following method was used to deliver tyr-RNP to edit melanocytes in the skin tissue of the leopard gill spiny perch:

[0051] (1) Fish selection: Select 4-month-old leopard gill spiny perch.

[0052] (2) Preparation before the experiment:

[0053] Preparation of the Cas9 / gRNA complex (RNP) targeting the tyr gene: 500 ng of gRNA dry powder was centrifuged for 10 min, dissolved in 20 μL TE Buffer (100 μM), and incubated at 4℃ for 30 min to adjust the final concentration to 25 μM. 10 μL of Cas9 protein stock solution (Alt-RspCas9 Nluclease V3 100 μg 62 μM) was mixed with 14.8 μL of Cas9 stock solution (20 mM HEPES-NaOH pH 7.5, 350 mM KCl, 20% glycerol, and enzyme-free water to 1 mL) to achieve a final concentration of 25 μM.

[0054] Mix 1 μL of cas9 protein (25 μM), 3 μL of gRNA (25 μM), and 1 μL of 1×PBS buffer, and incubate at room temperature for 15 min to obtain RNP.

[0055] (3) Electroporation Experiment Procedure: After anesthetizing the leopard-gill spiny perch with MS-222, the RNP substance to be delivered was dropped onto the surface of the fish using a precision syringe (Hamilton, USA). A roller with a microneedle length of 0.5 mm was used to perform 10 transverse rolling presses and 10 longitudinal rolling presses on the surface of the fish. Electroporation was then performed using a NEPA21 super electroporator and a CUY650P3 electrode. The following parameters were set: Poring Pulse: voltage 40 V, length 10 ms, interval 100 ms, 3 pulses, attenuation rate 10%, positive polarity; Transfer Pulse: voltage 20 V, length 75 ms, interval 50 ms, 5 pulses, attenuation rate 40%, alternating positive / negative polarity; 2 shocks were performed. A negative control group was also set up: electroporation was performed only (without RNP). On day 1 and day 5 after the experiment, the editing of melanocytes in the RNP delivery area was observed under a microscope, and the experimental group was compared with the negative control group. The results are shown in […]. Figure 3 .

[0056] Figure 3Figure 1 shows the bright-field morphology of melanocytes in the skin tissue of the leopard-gill spiny perch. Experimental groups A and B: Bright-field morphology of melanocytes on day 1 (A) and day 5 (B) after RNP delivery via electroporation. Arrows indicate the RNP-targeted editing sites, consistently showing a specific phenotype of significantly reduced melanocytes, while damage in surrounding non-targeted areas has recovered. Figure E is a magnified view of a portion of Figure B. Negative control groups C and D: Bright-field morphology of melanocytes on day 1 (C) and day 5 (D) after electroporation only (without RNP), showing complete recovery of damage. The scale bar for Figures A, B, C, D, and E is 250 μm.

[0057] The results showed that on day 1, thermal damage caused by the electrical pulse was observed under bright field imaging with a decrease in local melanocyte density. By day 5, the thermally damaged area had essentially recovered to a level of melanocyte density and morphology consistent with the surrounding normal skin. However, the RNP-targeted editing region consistently exhibited a specific phenotype of significantly reduced melanocytes, clearly identifiable under bright field imaging. The control group (electroporation only, without RNP) also showed thermal damage on day 1, but recovered to normal morphology by day 5, without any persistent changes in melanocytes. These results indicate that the thermal damage induced by this method is a transient and reversible process, while the RNP-mediated gene editing effect is stable in the tissue and can be visually identified through bright field morphological features.

[0058] Skin tissues from the experimental group and the negative control group of Leopard Gill Sea Bass were collected and identified by T7E1 enzyme digestion.

[0059] The specific methods are as follows: ①. Crude extraction of genomic DNA by alkaline lysis: Skin tissue from the electrocautery area of ​​the experimental group and the negative control group was taken, and 80 μL of alkaline lysis buffer (alkaline lysis buffer formula: 50 Mm NaOH, ddH2O to a final volume of 50 mL) was added. The mixture was incubated at 95℃ for 1 h, and 1 / 10 volume of 1 M Tris-HCl (pH 8.0) was added to neutralize the alkaline solution. The mixture was thoroughly mixed and centrifuged at maximum speed for 2–5 min. The supernatant was collected and stored at -20℃. ②. PCR amplification: The target gene was amplified for 30 cycles according to the following program: 98℃ for 10 min; 98℃ for 6 s; 57℃ for 15 s; 72℃ for 6 s; 72℃ for 10 min. The amplified gene was stored at 4℃. The PCR products were then subjected to renaturation in a PCR thermal cycler. The renaturation program was 95 °C for 5 min, followed by cooling from 95 °C to 85 °C at a rate of 2 °C per second, then from 85 °C to 25 °C at a rate of 0.1 °C per second, and finally held at 4 °C to form heteroduplex DNA. ③. T7E1 digestion: 8 μL of the renatured PCR product was added to 1 μL of sterile water, 2 μL of 1×NE Buffer 2, and 0.1 μL of T7 Endonuclease I, and incubated at 37 °C for 2 h for digestion. The digestion products were observed by gel electrophoresis, and the results are shown in the figure. Figure 4 The results of T7E1 enzyme digestion and agarose gel electrophoresis showed that the PCR products in the three parallel experimental groups showed the expected cleavage bands after enzyme digestion (indicated by red arrows), while the control group showed no cleavage bands.

[0060] As can be seen, the above experiment can successfully deliver tyr-RNP to edit melanocytes via electroporation.

Claims

1. A method of electroporation-mediated gene transfection or gene editing of fish skin tissue, characterized in that, The method comprises the following steps: After the fish body is anesthetized, the exogenous molecules are added dropwise to the surface of the fish body, and the microneedle roller is used to roll transversely and longitudinally on the surface of the fish body; then, electroporation is performed to deliver the exogenous molecules into the skin tissue of the fish body; The parameters of the electroporation are as follows: perforation pulse: voltage 40 V, length 10 ms, interval 100 ms, 3 pulses, decay rate 10%, positive polarity; transfer pulse: voltage 20 V, length 75 ms, interval 50 ms, 5 pulses, decay rate 40%, positive / negative alternating polarity; the number of electric shocks is 2.

2. The electroporation-mediated fish skin tissue gene transfection or gene editing method according to claim 1, characterized in that: The fish is Acanthoparectodus.

3. The electroporation-mediated fish skin tissue gene transfection or gene editing method according to claim 1, characterized in that: The exogenous molecule is either an EGFP plasmid or a Cas9 / gRNA complex targeting tyr the gene of interest.

4. The electroporation-mediated fish skin tissue gene transfection or gene editing method according to claim 1, characterized in that: The length of the microneedle of the microneedle roller is 0.4-0.6 mm, the number of transverse rolling is 8-12 times, and the number of longitudinal rolling is 8-12 times.

5. The electroporation-mediated fish skin tissue gene transfection or gene editing method according to claim 4, characterized in that: The length of the microneedle of the roller is 0.5 mm, the number of transverse rolling is 10 times, and the number of longitudinal rolling is 10 times.

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