A method for microinjection of shrimp and crab embryos, and a method for gene editing of shrimp and crabs

Abstract

The application discloses a kind of shrimp and crab class animal embryo microinjection method, shrimp and crab class animal gene editing method.The shrimp and crab class animal embryo microinjection method of the present application first obtains the fertilized egg embryo of shrimp and crab class animal in one cell stage, then using microinjection needle injects injection liquid into the fertilized egg embryo, wherein the inner diameter of the needle tip opening of microinjection needle is not more than 1 μm.The shrimp and crab class animal gene editing method of the present application uses the shrimp and crab class animal embryo microinjection method to complete microinjection, and then optimizes the culture conditions to improve the survival rate.The present application significantly improves the reliability and efficiency of the gene manipulation experiment on the embryos of Macrobrachium rosenbergii using the microinjection method, has important theoretical and practical significance for the research of functional genes of Macrobrachium rosenbergii and gene editing breeding, and also provides an important reference for the microinjection operation of embryos of other shrimp and crab crustaceans.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for microinjection into shrimp and crab embryos and a method for gene editing in shrimp and crabs. Background Technology

[0002] Giant freshwater prawn (Macrobrachium rosenbergii) Macrobrachium rosenbergii The giant freshwater prawn (Macrobrachium rosenbergii) is an important economic freshwater shrimp species widely distributed in tropical and subtropical regions. It possesses excellent characteristics such as rapid growth, large size, strong adaptability, and high aquaculture efficiency, thus being widely used in freshwater aquaculture worldwide. However, with the increasing prominence of problems such as germplasm degradation and frequent disease outbreaks caused by intensive farming, existing farmed populations are no longer able to meet the demands of high-quality industry development in terms of growth performance and stress resistance. Therefore, accelerating research on the breeding of superior varieties of giant freshwater prawns and cultivating new germplasm resources that are high-yielding, disease-resistant, and of high quality has become a key task for the sustainable development of the giant freshwater prawn industry.

[0003] While traditional breeding methods, such as phenotypic selection and empirical hybridization, have played a fundamental role in the early population improvement of *Macrobrachium rosenbergii*, these methods have significant limitations, including long breeding cycles, slow genetic progress, and low efficiency in the segregation and fixation of desirable traits. They are therefore insufficient to effectively address current industry problems such as germplasm degradation and high disease prevalence. With the increasing demand for efficient and precise breeding in aquaculture, precision breeding technologies relying on molecular genetic information are gradually becoming a key pathway to promote the sustainable development of the aquaculture seed industry. CRISPR / Cas9 gene editing technology, as a novel genome improvement tool, has been applied in the genetic breeding of many crops and some aquatic animals due to its ease of operation, high editing efficiency, and ability to precisely modify specific sites. However, its application in decapod animals such as shrimp and crabs remains relatively lagging, mainly due to the complexity of embryonic development, insufficient genomic resources, and the lack of a stable and efficient gene manipulation system.

[0004] Embryo microinjection, a precise and efficient method for introducing exogenous substances, plays an irreplaceable role in modern biological research and breeding applications. By directly injecting nucleic acid molecules, gene editing tools, or markers into fertilized eggs or early embryonic cells, microinjection can achieve gene overexpression, gene knockdown, gene deletion, and targeted modification, which can then be used for gene function analysis, genetic mechanism research, and the creation of novel germplasm. Particularly in species with limited genomic resources or incomplete gene manipulation systems, embryo microinjection provides crucial technical support for functional genomics research and precision breeding. Therefore, establishing an efficient embryo microinjection method for *Macrobrachium rosenbergii* is of significant scientific importance and promising application prospects for the precision molecular breeding and industrial upgrading of this species.

[0005] For example, the application with publication number CN108070617A discloses a method for microinjection of Exopalaemon modestus embryos and a method for constructing mRNA overexpression model. The method is to perform microinjection on Exopalaemon modestus single-cell stage embryos under high osmotic pressure microinjection system and specific microinjection conditions. The related conditions of microinjection include the pressure selected during microinjection: the pressure should not exceed 80 Pa, otherwise the internal material arrangement of the zygote will be disturbed; the amount of microinjection: the amount of microinjection into the zygote should be moderate, too little is not enough for gene editing, and too much is harmful to the zygote. The application adopts an injection amount of 0.5 nL; the time of microinjection: the time of microinjection is positively correlated with the amount of microinjection, therefore, in order to ensure a certain amount of microinjection, the time of microinjection should not exceed 0.7 s; the culture environment of the zygote after microinjection: incubated in fresh seawater filtered by 0.22 μm filter membrane.

[0006] However, the yolk content of Macrobrachium rosenbergii zygote is rich, almost fills the whole zygote, and microinjection can easily cause the zygote to be damaged and burst, and the embryo after injection is difficult to hatch, so that the success rate of successfully culturing the genetically edited shrimps after microinjection of zygote embryo is low. SUMMARY

[0007] The present application provides a shrimp and crab animal embryo microinjection method and a shrimp and crab animal gene editing method to solve the above problems in the prior art.

[0008] The present application first provides a shrimp and crab animal embryo microinjection method, which comprises the following steps:

[0009] S1, obtaining a zygote embryo of a shrimp and crab animal in one-cell stage;

[0010] S2, injecting an injection liquid into the zygote embryo by using a microinjection needle, wherein the inner diameter of the needle tip opening of the microinjection needle is not greater than 1 μm.

[0011] Preferably, the shrimp and crab animal is a Macrobrachium animal. More preferably, the shrimp and crab animal is Macrobrachium rosenbergii.

[0012] Preferably, in step S1, the zygote embryo in one-cell stage is half to three hours after fertilization.

[0013] In step S1, the embryo mass on the female Macrobrachium rosenbergii half to three hours after fertilization is removed, and the embryos are dispersed by using trypsin for digestion. After digestion, single particle embryos with complete shape, smooth surface and no rupture are picked for microinjection.

[0014] Preferably, the effective length of the needle tip of the microinjection needle is 8-9 mm. For the microinjection needle, the opening generally used in the prior art is relatively large. The material of the microinjection needle is glass, that is, the microinjection needle is an elongated glass tube, and the elongated needle tip can be obtained by further needle drawing after heating. The longer the needle tip, the smaller the opening can be. If the effective length of the needle tip is insufficient, the opening of the needle tip is not easy to accurately operate when being polished after heating and needle drawing, and the opening of the needle tip is easily too large due to excessive polishing.

[0015] Preferably, in step S2, the microinjection amount in each fertilized egg embryo is 0.5 nL, the injection pressure is 200-300 kPa, and the injection time is 0.2 s.

[0016] The application further provides a shrimp and crab animal gene editing method, comprising the following steps:

[0017] (1) using the shrimp and crab animal embryo microinjection method to obtain the microinjected fertilized egg embryo, wherein the injection solution injected into the fertilized egg embryo is a reagent for gene editing;

[0018] (2) culturing the microinjected fertilized egg embryo in a half-seawater solution with a mass concentration of 6.5‰ on the first day to develop the fertilized egg embryo from the cleavage stage to the blastula stage;

[0019] (3) culturing in a full-seawater solution containing 1%-10% polyethylene glycol with a mass concentration of 13‰ from the second day to the fourth day to develop the fertilized egg embryo from the blastula stage to the gastrula stage;

[0020] (4) continuing to culture in the full-seawater solution with a mass concentration of 13‰ until hatching out of the membrane.

[0021] Preferably, in step (2), oscillation is performed during the culturing process, the rotation speed is 20 rpm, the temperature is controlled at 28℃, and the light:dark cycle condition is 14:10 hours.

[0022] Preferably, in step (3), the mass concentration of the polyethylene glycol is 1%-4%, preferably 2%-3%, and most preferably 2%.

[0023] Preferably, the molecular weight of the polyethylene glycol is 1500-6000, preferably 3000-4000.

[0024] Compared with the prior art, the application has the following advantages:

[0025] This invention overcomes key problems in giant freshwater prawns, such as egg-bearing, highly adhesive fertilized eggs, hard egg membranes, easy rupture upon puncture, and high mortality rate after injection. This invention significantly improves the reliability and efficiency of gene manipulation experiments on giant freshwater prawn embryos using microinjection. It has important theoretical and practical significance for research on functional genes in giant freshwater prawns and gene editing breeding, and also provides an important reference for microinjection operations on embryos of other crustaceans such as shrimp and crabs. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of injection using a microinjection needle in an embodiment of the present invention. The red arrows indicate the injected solution that is diffusing.

[0027] Figure 2 The images show the injection effect of the microinjection needle used in the embodiments and comparative examples of this invention.

[0028] Figure 3 Wild-type WT and different injection batches of giant freshwater prawn embryos MrPAX6 Image showing the sequencing results of gene PCR products.

[0029] Figure 4 For the corresponding Figure 3 Different batches of giant freshwater prawn embryos injected MrPAX6 Histogram of frequencies of various insertion and deletion mutation types in genes.

[0030] Figure 5 Day 8 (8 dpf) and day 14 (14 dpf) after injection of giant freshwater prawn embryos MrPAX6 Phenotypic comparison diagram of gene mutant and wild-type WT. Detailed Implementation

[0031] Example 1

[0032] 1. Prepare the embryos for injection:

[0033] The premise of microinjection is to obtain one-cell stage embryos of fertilized eggs, while the natural mating of prawns and crabs usually occurs at night, which greatly affects the development of microinjection. To solve this problem, we have explored a scheme for indoor daytime mating and spawning of Macrobrachium rosenbergii, which can obtain a large number of one-cell stage embryos for microinjection every day. At around 6:00 in the morning on the day of injection, female prawns that have just molted and have full gonads but have not laid eggs, and male prawns with intact claws and strong vitality, are placed in a small mating pool at 28°C in a constant temperature and oxygenated indoor environment, with a female to male ratio of 1:2. The female prawns are observed every hour for egg-laying. Generally, after 6 hours of mating, the female prawns will lay eggs, and one-cell stage embryos of M. rosenbergii can be obtained between 14:00 and 18:00 in the afternoon. Based on experience, freshly laid fertilized eggs are very fragile and can be broken with a single touch. The egg membranes become hard after the two-cell and four-cell stages, making injection difficult. The best injection window period is usually within half an hour to three hours after fertilization.

[0034] M. rosenbergii is a clutch-hatching crustacean, and its fertilized eggs are in the form of clumps adhering to the mother's appendages on the abdomen. In order to carry out microinjection experiments, these clumps of fertilized eggs need to be processed into single, dispersed embryo particles. The following are the specific steps for obtaining single embryos:

[0035] Select M. rosenbergii fertilized eggs that have been fertilized for more than 0.5 hours. At this time, the embryos have not begun to develop significantly, and their morphological structures are intact, making them suitable for subsequent processing.

[0036] Carefully remove the embryo clumps with tweezers and collect them in a clean centrifuge tube, avoiding excessive pressure to prevent damage to the embryos.

[0037] Prepare a 1 mg / mL concentration of trypsin solution (use a 6.5‰ half-seawater solution as the solvent, and the temperature is 28°C).

[0038] Add an appropriate amount of trypsin solution to the collected embryo clumps, and place them at room temperature (about 25-28°C). Gently shake and oscillate to ensure that the trypsin acts uniformly on the surface of the embryos, and continue for 5-15 minutes.

[0039] After the embryos are completely dispersed, quickly transfer the embryo solution to a new centrifuge tube and add an equal volume of 6.5‰ half-seawater solution containing 1% fetal bovine serum (FBS) to terminate the activity of trypsin. Replace the fresh 6.5‰ half-seawater solution twice to completely remove the residual trypsin and prevent it from continuing to act and causing damage to the embryos.

[0040] Under a stereomicroscope, select single particle embryos with complete morphology, smooth surface, and no rupture, and transfer them to a 3% agarose groove for injection, with a groove depth of about 0.5 mm and a length of about 5 cm per row. This allows the embryos to be fixed and arranged neatly, facilitating microinjection operations.

[0041] 2. Preparation of microinjection needle:

[0042] In a large number of practical operations, we found that the specification and opening of the microinjection needle are crucial to the survival rate of the fertilized eggs of Macrobrachium rosenbergii. The internal osmotic pressure of the fertilized eggs of Macrobrachium rosenbergii is high, and the egg membrane will gradually harden after fertilization. Therefore, if the injection needle is too short or the opening is too wide, the fertilized eggs will burst and die in large numbers. If the injection needle is too long or the opening is too small, the needle will be difficult to penetrate the egg membrane, and injection will be difficult. Therefore, we have carried out a large number of optimizations. The present application provides a Macrobrachium rosenbergii embryo microinjection needle with strong penetration, low trauma, and high survival rate after injection. The specific steps are as follows:

[0043] Experimental apparatus and material preparation: glass capillary, outer diameter 1.00 mm, inner diameter 0.50 mm, transparent high-quality borosilicate glass (such as Sutter or Narishige brand capillary), the tube wall should be smooth and crack-free before use, and can be wiped with alcohol cotton to remove dust if necessary; program-controlled horizontal needle drawing instrument HL-1000, check whether the heating wire is complete and the needle drawing slide rail is clean and unobstructed before starting; needle grinding instrument PG-22C, used for fine grinding of the drawn glass needle to ensure that the needle tip is sharp and the opening is moderate to penetrate the shrimp egg membrane without damaging the internal structure of the embryo.

[0044] Needle drawing operation steps: carefully place the glass capillary vertically into the clamp of the needle drawing instrument, ensuring that the two ends are centered and symmetric, and fix it slightly to prevent sliding or breaking during the drawing process; set the needle drawing parameters, heating temperature 500℃, needle drawing force 50N, needle drawing speed 150mm / s, air pump air pressure 300kPa (for cooling or rapid clamping), heating time 120ms, cycle number 1, start the program, the instrument will automatically complete the heating softening and rapid stretching of the glass tube; after the drawing is completed, take out the two symmetric needles and observe the needle tip shape under a stereomicroscope to confirm that the needle tip is stretched evenly and has no burrs.

[0045] Grinding the injection needle steps: gently insert one of the drawn glass needles into the fixed clamp of the needle grinding instrument, with the needle tip facing down and maintaining an appropriate distance from the grinding disc surface (contact but not pressed tightly), and pay attention to keep the needle tip angle consistent to avoid bending or deviation; set the grinding parameters, the rotation speed is set to 2100rpm, the grinding angle is 30°, and the grinding time is ≥1 minute (which can be adjusted according to the desired opening); check the opening size of the ground needle tip under a stereomicroscope or high-power microscope, the effective length of the needle tip is about 8-9mm; the inner diameter of the needle tip opening is about 1μm, and in the ideal state it should be a regular circle or ellipse with sharp edges and no burrs; the ground injection needle should be avoided from contacting with hard objects, and can be inserted into a dust-free foam board or placed on a special needle rack for temporary storage.

[0046] 3. Preparation of microinjection samples:

[0047] The application of this invention mainly targets genes that regulate eye development in giant freshwater prawns. MrPAX6 Embryo-level editing is performed. The injection material is Cas9 protein and a target... MrPAX6 The guide sgRNA and Cas9 protein were purchased from NEB (catalog number: M0646T, concentration: 20 μM). The target site sequence of the sgRNA was 5'-TCTCGCGGGCAAATACGTCA-3'. The full-length sgRNA sequence was synthesized by Nanjing Genscript Biotech Co., Ltd. at a concentration of 10 μM. Both Cas9 protein and sgRNA were stored at -80℃. Before injection, Cas9 protein and sgRNA were taken out and mixed evenly at a molar ratio of Cas9:sgRNA = 1:1.6 to form an RNP mixture. Then, 1~2 μL of the RNP mixture was loaded into the prepared microinjection needle using a micropipette tip.

[0048] 4. Microinjection and post-injection embryo hatching:

[0049] The microinjection needle loaded with Cas9 / sgRNA solution was fixed to the microsyringe connection system, and an injection pressure test was performed to ensure that the needle tip was unobstructed and the injection was stable. Using the microinjection needle obtained by this invention, the required injection pressure is 200-300 kPa, the injection time is 0.2 s, and the output volume is about 0.5 nL. Under the microscope, the injection needle was slowly advanced to pierce the egg membrane and enter about 1 / 4 of the embryo's diameter. The injection pressure pedal was pressed to inject the mixture, and the injection needle was withdrawn.

[0050] The microinjection method described in this embodiment can successfully inject exogenous solutions into one-cell stage fertilized eggs of Giant freshwater prawns. The microinjection needle has strong penetration and causes no obvious damage to the fertilized eggs. Figure 1 Survival and mortality were recorded 24 hours later. Compared with Comparative Example 1 (injection using a microinjection needle with an opening of approximately 1.5 μm and a tip size of 8-9 mm), Comparative Example 2 (opening of approximately 2 μm and a tip size of 7-8 mm), Comparative Example 3 (opening of approximately 2.5 μm and a tip size of 7-8 mm), and Comparative Example 4 (opening of approximately 3 μm and a tip size of 7-8 mm), the microinjection method used in this embodiment can significantly improve the survival rate of Macrobrachium rosenbergii embryos after microinjection. Figure 2 Table 1 lists the survival rates of different batches of injections under different protocols. It can be seen that the smaller the opening of the injection needle, the higher the survival rate. Furthermore, when the opening is increased to 1 μm, the survival rate is significantly improved, from less than 10% to about 90%.

[0051] Table 1

[0052]

[0053] The injected embryos are extremely fragile, and good hatching conditions are one of the keys to ensure normal development. Generally, the first day is the cleavage stage, and the internal osmotic pressure does not change much. The embryos are cultured in a 10 cm culture dish at a density of 100 per 15 mL of culture solution with a salinity of 6.5 ‰, and the culture dish is placed on a horizontal shaking table at a speed of 20 rpm to ensure that the water is slightly flowing, the embryos are not deposited and adhered, and the temperature is controlled at 28 ℃, and a light:dark cycle of 14:10 hours is provided. From the second day to the fourth day, the embryos of Macrobrachium rosenbergii develop from the blastula stage to the gastrula stage, and the intracellular osmotic pressure changes greatly. The mortality rate of the injected embryos begins to gradually rise, and the embryos are cultured in a 13 ‰ full seawater solution containing 2% (g / mL) PEG4000, which can significantly improve the survival rate of the embryos. PEG has a certain water absorption, and a layer of molecular film is formed on the surface of the cells after contacting with the cells, so that the cells are in a state of mild dehydration. Compared with ordinary seawater, the growth of the hyphae on the surface of the embryos during hatching can be effectively inhibited, and the osmotic pressure can be maintained.

[0054] Compared with the use of a 6.5 ‰ half seawater solution or a 13 ‰ full seawater solution throughout the hatching process, the survival rate of the injected embryos during the hatching process can be greatly improved by using the hatching process of the present embodiment. Table 2 lists the survival rates of different batches of embryos hatched to the 14th day under different schemes, as shown in Table 2.

[0055] Table 2

[0056]

[0057] In addition, we also explored the effect of 2% PEG (PEG1500, PEG3000, PEG4000, PEG5000, PEG6000) with different polymerization degrees added to a 13 ‰ full seawater solution on the survival rate of the injected embryos during hatching. The results are shown in Table 3. It can be seen that when the polymerization degree changes from low to high, the survival rate first increases and then decreases, and the survival rate is the highest when PEG4000 is added.

[0058] Table 3

[0059]

[0060] In addition, we also explored the effect of PEG4000 with different concentrations (g / mL) added to a 13 ‰ full seawater solution on the survival rate of the injected embryos during hatching. The results are shown in Table 4. It can be seen that when the concentration increases from 1% to 2%, the survival rate increases from more than 30% to about 50%, but when the concentration continues to increase, the survival rate gradually decreases.

[0061] Table 4

[0062]

[0063] 5. Phenotype observation and mutation detection of injected individuals:

[0064] At 48 h after injection, 10 surviving embryos were taken to extract genomic DNA using the "animal tissue genomic extraction kit" of Kangwei Century Company. Primers located on both sides of the target site were designed, in which MrPAX6 F is 5'-TGGGTCTTACCCTTGATCCT-3', MrPAX6 R is 5'-AGTAACAATGAACGTGACGC-3', and genomic DNA was used as a template for PCR amplification. The PCR reaction system is shown in Table 5.

[0065] Table 5

[0066]

[0067] The PCR reaction program is set as follows: initial denaturation at 95℃ for 3 minutes, followed by 30 cycles, each cycle including denaturation at 95℃ for 30 seconds, annealing at 58℃ for 30 seconds and extension at 72℃ for 20 seconds, and a final extension step of 72℃ for 10 minutes. After 1.5% agarose gel electrophoresis analysis of the PCR amplification product, sequencing was performed, and three independent repeated experiments showed that the peaks were nested or interrupted near the target site ( Figure 3 ), indicating that the MrPAX6 gene of the Macrobrachium rosenbergii embryo was successfully edited. The frequency of base insertion and deletion in gene editing was calculated using TIDE software, and the results showed that the gene editing efficiency was 46.9%, 39.6% and 50% respectively ( Figure 4 ).

[0068] On the 8th day (8dpf) and the 14th day (14dpf) after embryo injection, the survival state and eye spot development of the embryos were observed under a stereomicroscope, and images were collected. The observation results are shown in Figure 5 , in which "WT" is the wild type individual, and "MrPAX6" is the mutant obtained after knocking out the target gene MrPAX6 . The experimental results show that MrPAX6 gene knockout leads to obvious eye spot development defects, and the eye spot size of the mutant individual is about 1 / 4 of the wild type, with obvious phenotype difference.

Claims

1. A method of gene editing in a shrimp / crab animal, comprising, The method comprises the following steps: (1) obtaining a microinjected fertilized egg embryo by using a shrimp and crab embryo microinjection method, wherein the injection solution injected into the fertilized egg embryo is a reagent for gene editing; The shrimp and crab embryo microinjection method comprises the following steps: S1, obtaining a fertilized egg embryo of a shrimp and crab in a one-cell stage; S2, injecting an injection solution into the fertilized egg embryo by using a microinjection needle, wherein the inner diameter of the needle tip opening of the microinjection needle is 1 μm; and the effective length of the needle tip of the microinjection needle is 8-9 mm; The shrimp and crab is Dendrobaena; (2) culturing the microinjected fertilized egg embryo in a half-seawater solution with a mass concentration of 6.5‰ for the first day to develop the fertilized egg embryo from the cleavage stage to the blastula stage; (3) culturing in a full-seawater solution containing 1%-4% polyethylene glycol with a mass concentration of 13‰ from the second day to the fourth day to develop the fertilized egg embryo from the blastula stage to the gastrula stage; the molecular weight of the polyethylene glycol is 3000-4000; (4) continuing to culture in the full-seawater solution with a mass concentration of 13‰ until hatching out of the membrane.

2. The gene editing method for shrimp and crab animals according to claim 1, characterized in that, In step (2), the culture process is oscillated at a speed of 20 rpm; the temperature is controlled at 28℃, and the light:dark cycle is 14:10 hours.

3. The gene editing method for shrimp and crab animals according to claim 1, characterized in that, The mass concentration of the polyethylene glycol is 2%-3%.

4. The gene editing method for shrimp and crab animals according to claim 1, characterized in that, In step S1, the fertilized egg embryo in the one-cell stage is half an hour to three hours after fertilization; In step S1, the embryo mass on the female Dendrobaena half an hour to three hours after fertilization is removed, digested by using trypsin to disperse the embryos, and after digestion, single particle embryos with complete shape, smooth surface and no rupture are picked for microinjection.

5. The gene editing method for shrimp and crab animals according to claim 1, characterized in that, In step S2, the microinjection amount in each fertilized egg embryo is 0.5 nL, the injection pressure is 200-300 kPa, and the injection time is 0.2 s.

Images