A fusion protein PmMAD7-NLS for gene editing of Penaeus monodon and its application

Abstract

The invention discloses a fusion protein PmMAD7-NLS for gene editing of Penaeus monodon. The nucleotide sequence encoding the fusion protein PmMAD7-NLS is shown in SEQ ID NO: 4. The invention also discloses a nucleotide construct comprising the fusion protein PmMAD7-NLS, and application of the fusion protein PmMAD7-NLS or the nucleotide construct in site-directed editing of the Penaeus monodon genome. The fusion protein PmMAD7-NLS improves the expression efficiency and stability of MAD7 in Penaeus monodon cells through codon optimization technology, thereby significantly improving the accuracy and efficiency of gene editing. The fusion protein PmMAD7-NLS is combined with a nuclear localization signal NLS to enhance the localization of the codon-optimized MAD7 in the cell nucleus.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering and gene editing technology, and specifically relates to a method for Penaeus monodon ) Fusion proteins for genome site-specific editing and their applications. Background Art

[0002] Gene editing technology, especially the application of CRISPR-Cas system, has become a key technology in modern biological science and biotechnology. Among them, MAD7 nuclease, as an emerging CRISPR tool, has unique structural and functional properties. MAD7 is a new type of CRISPR system-related nuclease developed by Inscripta, USA, and belongs to the MADzymes family named after the biodiversity-rich Madagascar Island. The discovery of MAD7 originated from the exploration of different CRISPR systems, aiming to expand the application range of nucleic acid-guided endonucleases and improve the diversity of PAM recognition sequences, cutting efficiency, enzyme size and kinetic performance (Liu et al., 2020).

[0003] MAD7 is a 1263 amino acid protein that shows homology to the Cas enzyme family of type V (Cpf1-like) CRISPR systems. It is structurally similar to Cpf1 from Acidaminococcus sp., and although the overall amino acid sequence similarity is only 31%, the sequence of the PAM recognition domain is highly conserved. MAD7 uses the YTTNPAM site when designing gRNA, a finding based on its application in Escherichia coli (E. coli) and Saccharomyces cerevisiae (S. cerevisiae), where approximately 95% of genomic locations are within 50 nucleotides of a YTTN site (Vanegas et al., 2022).

[0004] In addition to its application in microbial systems, MAD7 has also demonstrated its potential in more advanced organisms. For example, in plant models such as wheat and rice, MAD7 successfully achieved efficient editing of specific genes TaDEP1, TaVRN1, OsALS-T2, OsEPSPS-T1, and OsNRAMP5-T1. The editing frequencies of TaDEP1 and TaVRN1 in wheat were 1.5% and 3.0%, respectively, while the mutation frequencies in the OsALS-T2, OsEPSPS-T1, and OsNRAMP5-T1 genes of rice were 49.0%, 65.6%, and 56.9%, respectively. The mutation frequency is significant, demonstrating its application value in agricultural biotechnology (Lin et al., 2021). Similarly, in the zebrafish model, MAD7 successfully mediated efficient and precise gene editing through precisely designed crRNA and optimized nucleic acid delivery system, combined with the strategy of heat shock treatment to enhance the activity of the CRISPR system (Price et al., 2020).

[0005] Although MAD7 and other CRISPR systems have achieved remarkable results in the application of microorganisms, plants and model animals, their application in marine organisms such as crustaceans such as Penaeus monodon still faces many challenges. The genome of Penaeus monodon is relatively large and complex, with a large number of repetitive sequences and a high proportion of non-coding DNA, which increases the difficulty of designing gRNA during gene editing, because suitable PAM recognition sites are relatively scarce, which may affect the accuracy and efficiency of editing. In addition, as an aquatic animal, the cell structure in the body of Penaeus monodon is different from that of terrestrial organisms, and the existing nucleic acid delivery methods are not ideal in this species, especially the exoskeleton and skin barriers hinder effective nucleic acid delivery. In addition, the immune system of Penaeus monodon may produce a strong immune response to the CRISPR system, reducing the efficiency of gene editing. Moreover, the special physiological conditions in the marine environment, such as salinity, temperature and pH, may affect the stability and effectiveness of the CRISPR system, thereby limiting the editing efficiency. In addition, Penaeus monodon lacks an effective gene editing screening and detection system, which further increases the complexity of technical application.

[0006] To address these issues, future research needs to focus on developing nucleic acid delivery methods that are more suitable for marine organisms, optimizing the editing efficiency of the CRISPR system, and establishing an efficient gene screening and detection system. As an emerging CRISPR tool, MAD7 shows the potential to overcome the limitations of existing technologies. Its PAM recognition sequence has greater flexibility and can find more suitable editing sites in complex genomes, especially in species with large genomes such as Penaeus monodon. In addition, the structure and small protein size of MAD7 make it easier to apply to these marine organisms through optimized delivery systems. At the same time, because it is derived from the CRISPR system of non-model organisms, it may reduce the immune rejection of Penaeus monodon and further improve the editing efficiency. Therefore, MAD7 is expected to become an effective tool to solve the problem of gene editing in Penaeus monodon through these improvements. Summary of the invention

[0007] The present invention aims to provide a fusion protein PmMAD7-NLS for gene editing of Penaeus monodon and a nucleotide construct comprising the fusion protein PmMAD7-NLS.

[0008] The present invention also aims to provide the use of the above fusion protein PmMAD7-NLS or the nucleotide construct in site-directed editing of the Penaeus monodon genome.

[0009] The above-mentioned first object of the present invention can be achieved by the following technical scheme: a fusion protein PmMAD7-NLS for gene editing of Penaeus monodon, and the nucleotide sequence encoding the fusion protein PmMAD7-NLS is shown in SEQ ID NO: 4.

[0010] Preferably, the fusion protein PmMAD7-NLS is a codon-optimized CRISPR-associated endonuclease modified with the nuclear localization signal NLS.

[0011] Preferably, the PmMAD7 is a codon-optimized coding sequence of the CRISPR-associated endonuclease MAD7, the nucleotide sequence encoding the CRISPR-associated endonuclease MAD7 is shown in SEQ ID NO: 1, the amino acid sequence of the CRISPR-associated endonuclease MAD7 is shown in SEQ ID NO: 2, and the nucleotide sequence encoding the PmMAD7 is shown in SEQ ID NO: 3.

[0012] The fusion protein PmMAD7-MLS (referred to as NLS-PmMAD7-NLS in the examples below) is a codon-optimized CRISPR-associated endonuclease MAD7 modified with a nuclear localization signal (NLS), which enhances localization and activity in the nucleus of Penaeus monodon cells.

[0013] Preferably, the nuclear localization signal NLS is located at the N-terminus and C-terminus of the CRISPR-associated endonuclease MAD7 after codon optimization, and is composed of nuclear localization signals derived from SV40 and nucleoplasmin, respectively.

[0014] MAD7 is a new type of CRISPR-associated endonuclease with good gene editing potential. In order to optimize the activity of MAD7 in Penaeus monodon cells, the present invention adds NLS sequences to the N-terminus and C-terminus of codon-optimized MAD7, and the NLS sequences are respectively from SV40 and nucleoplasmin to enhance the localization of codon-optimized MAD7 in the cell nucleus.

[0015] Therefore, in order to improve the expression efficiency in Penaeus monodon cells, the present invention performs codon optimization on the coding sequence of MAD7 to match the gene translation mechanism of Penaeus monodon, thereby significantly improving the expression level of the fusion protein.

[0016] In addition, the present invention provides an efficient and accurate gene editing tool for genetic improvement of aquatic animals by performing codon optimization on the coding sequence of MAD7 specifically for Penaeus monodon and integrating the codon-optimized MAD7 nuclease into the nuclear localization signal (NLS).

[0017] The present invention also provides a nucleotide construct for expressing the fusion protein, wherein the nucleotide construct comprises a nucleotide sequence encoding the fusion protein PmMAD7-NLS.

[0018] Furthermore, the nucleotide construct for expressing the fusion protein of the present invention also includes a promoter and a terminator to achieve efficient expression of the nucleotide sequence in Penaeus monodon cells.

[0019] Preferably, the promoter is a strong promoter, such as CMV or T7, to ensure high-level expression in Penaeus monodon cells.

[0020] The present invention constructs a nucleotide construct containing the coding sequence of the fusion protein PmMAD7-NLS, and uses a strong promoter and terminator suitable for Penaeus monodon cells to ensure efficient expression of the fusion protein in the cell. Through this construct, the present invention can induce DNA double-strand breaks at the target gene site and perform precise genome editing through the cell's DNA repair mechanism.

[0021] Therefore, in terms of design, the present invention first optimizes the codons of Penaeus monodon, and fuses the NLS sequences of SV40 and nucleoplasmin to the N-terminus and C-terminus of the codon-optimized MAD7 nuclease, respectively, to adapt to the translation mechanism of its cells. The optimized expression vector contains a nucleotide construct of the PmMAD7-NLS coding sequence and a promoter and terminator suitable for expression in Penaeus monodon cells, thereby ensuring efficient protein production.

[0022] The above second object of the present invention can be achieved by the following technical solution: application of the above fusion protein or the above nucleotide construct in site-specific editing of the Penaeus monodon genome.

[0023] Further, the following steps are included:

[0024] (1) introducing the nucleotide construct into Penaeus monodon cells;

[0025] (2) Using the CRISPR-Cas system to induce double-stranded DNA breaks at the target gene site using the fusion protein PmMAD7-NLS;

[0026] (3) Achieve site-directed mutation, insertion or modification of genes through intracellular DNA repair mechanisms.

[0027] Therefore, in terms of application, the present invention uses the fusion protein PmMAD7-NLS to perform site-specific editing of specific genes guided by the CRISPR-Cas system, such as mutation, insertion or modification, which is suitable for gene function research and genetic improvement of shrimp. This efficient gene editing tool can not only improve the operability and success rate of gene editing, but also promote the healthy development and disease prevention and control of the shrimp farming industry. The fusion protein PmMAD7-NLS of the present invention provides an efficient and accurate new method for the gene editing field of Penaeus monodon through the dual enhancement strategy of codon optimization and nuclear localization signal, which has broad application prospects and practical value.

[0028] In general, in Penaeus monodon, the present invention enhances the localization and activity of codon-optimized MAD7 in the animal cell nucleus by fusing it to a nuclear localization signal (NLS), thereby improving the efficiency and accuracy of gene editing. In this way, codon-optimized MAD7 not only provides a new and efficient tool for gene editing, but is also particularly suitable for application scenarios that have special requirements for vector size and editing efficiency, such as genetic manipulation and disease control in complex aquatic organisms. These advances demonstrate the potential for widespread application of codon-optimized MAD7 in the field of gene editing, especially in the demanding aquaculture industry.

[0029] Therefore, the fusion protein PmMAD7-NLS or the above-mentioned nucleotide construct of the present invention can also be further used in improving the disease resistance, growth rate or meat quality of Penaeus monodon, in the genetic improvement of Penaeus monodon, and in improving the aquaculture benefits by optimizing the genetic characteristics of Penaeus monodon.

[0030] The present invention has the following advantages:

[0031] (1) High efficiency and precision: The present invention significantly improves the localization of MAD7 nuclease in the nucleus of Penaeus monodon cells by introducing nuclear localization signals (NLS) at both ends of the codon-optimized MAD7 nuclease. This optimized design not only enhances the activity of the enzyme, but also ensures efficient cutting ability during genome editing. Combined with the specific guiding ability of the CRISPR system, the present invention achieves high-precision gene editing and minimizes non-target effects and off-target risks.

[0032] (2) Codon optimization: Optimizing the codon preference of Penaeus monodon is one of the key innovations of the present invention. By optimizing the coding sequence of MAD7 and matching the translation mechanism of Penaeus monodon cells, the translation efficiency of mRNA and the expression level of protein (no expression before optimization, edited expression after optimization) were significantly improved. This codon optimization not only increased protein production, but also improved the stability and functional activity of MAD7 in Penaeus monodon cells, ensuring the consistency and repeatability of the editing effect.

[0033] (3) Adaptability and flexibility: The nucleic acid construct of the present invention is flexible in design and can be customized according to different target genes (such as ECH1) and application requirements. By adjusting the sequence of the guide RNA (gRNA) and the regulatory elements in the nucleic acid construct, the present invention can adapt to a variety of gene editing tasks, thereby achieving diversified genetic modification. This flexibility makes it have a wide range of application potential in aquaculture and can meet the needs of different species and production goals.

[0034] (4) Economic benefits and environmental friendliness: This invention reduces the time and cost investment in the testing and production process by improving the efficiency and accuracy of gene editing. Compared with traditional breeding and improvement methods, gene editing can achieve target characteristics faster and reduce production costs. At the same time, improved aquatic animals can reduce dependence on chemical drugs and antibiotics, reduce environmental pollution risks, and promote environmentally friendly and sustainable farming practices.

[0035] (5) Innovation-driven industrial development: This invention promotes the innovative application of CRISPR technology in aquaculture and stimulates the potential for transformation of new technologies in the industry. By introducing efficient gene editing tools, aquaculture companies can accelerate the development and industrialization of new varieties, thereby improving market competitiveness and product added value.

[0036] Therefore, the present invention provides an efficient, accurate and economical solution for gene editing of aquatic animals by combining NLS positioning and codon optimization technology, which has significant scientific value and commercial application prospects. Its wide adaptability and flexibility enable it to meet various genetic improvement needs and contribute to the sustainable development of aquaculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The present invention will be further described below in conjunction with embodiments with reference to the accompanying drawings.

[0038] Figure 1 Schematic diagram of PmMAD7 vector construction and verification in Example 1, respectively PmMAD7 vector construction, PmMAD7 crRNA structure analysis and PmMAD7 mRNA structure;

[0039] Figure 2 The PmMAD7-EGFP and PmMAD7-NLS-EGFP in Example 1 were transfected into Penaeus monodon, and the nauplii were observed by fluorescence;

[0040] Figure 3 It is the knockout verification of PmMAD7 in the blood cells of Penaeus monodon in Example 1, the peak diagram of the first generation sequencing of ECH1 target sequencing detection, ech-1, ech-2, and ech-3 all represent mutants (heterozygotes);

[0041] Figure 4 This is the peak diagram of the first generation sequencing of ECH1 target sequencing detection before MAD7 codon optimization in Example 1. DETAILED DESCRIPTION

[0042] The present invention is further described below in conjunction with specific examples. The following examples and drawings are only used for illustrative purposes and are not to be construed as limiting the present invention. Unless otherwise specified, the reagent raw materials used in the following examples are conventional commercially available or commercially available raw reagent raw materials, and unless otherwise specified, the methods and equipment used in the following examples are conventional methods and equipment used in the art.

[0043] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0044] Example

[0045] The present invention is further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0046] This embodiment of the present invention designs a fusion protein and a corresponding nucleic acid construct for efficiently editing the genome of Penaeus monodon. The specific implementation method includes the following steps:

[0047] 1. Construction of PmMAD7-NLS fusion vector

[0048] Vector design: The nuclear localization signal (NLS) was connected to the N-terminus and C-terminus of the codon-optimized MAD7 nuclease to enhance its localization and editing activity in the nucleus of Penaeus monodon cells. The NLS sequence of SV40 large T antigen and the NLS sequence of nucleoplasmin were selected, and the corresponding pattern Figure 1 , first codon optimization, and then connect the nuclear localization signal NLS.

[0049] 2. Construction of nucleic acid expression vector

[0050] 2.1 Nucleic acid sequence design and optimization

[0051] The coding sequence of MAD7 was codon-optimized to improve the expression efficiency of the protein in target cells.

[0052] The nucleotide sequence of MAD7 in humans before optimization is shown in SEQ ID NO: 1.

[0053] Its amino acid sequence is shown in SEQ ID NO: 2, and is as follows:

[0054]

[0055] The codon preference of Penaeus monodon was optimized without changing the corresponding amino acid sequence, and it was named PmMAD7. It was synthesized by artificial sequence synthesis. The nucleotide sequence encoding PmMAD7 is shown in SEQ ID NO: 3, which is as follows:

[0056]

[0057] Nuclear localization signals are connected to the N-terminus and C-terminus of the optimized MAD7 nuclease to enhance its localization and editing activity in the nucleus of Penaeus monodon cells. The NLS sequence of SV40 large T antigen and the NLS sequence of nucleoplasmin are selected, and the nucleic acid sequence encoding the fusion protein PmMAD7-NLS is shown in SEQ ID NO: 4, and is specifically as follows:

[0058]

[0059] 2.2 Vector construction

[0060] The NLS-PmMAD7-NLS nucleic acid sequence synthesized in 2.1 is inserted into a strong promoter (such as CMV or T7) and a terminator in the construct to ensure high-level expression in Penaeus monodon cells. Specifically, it is inserted into a GS-CMV vector through molecular cloning technology to ensure that the vector contains a strong promoter (such as CMV) to promote high-level expression.

[0061] 2.3 Vector construction and verification

[0062] Restriction enzyme digestion and PCR amplification were used to verify the correct insertion of the NLS-PmMAD7-NLS sequence into the vector.

[0063] The integrity and correctness of the inserted sequence were confirmed by sequencing.

[0064] 2.4 Transcription into NLS-PmMAD7-NLS mRNA

[0065] In vitro transcription

[0066] Perform in vitro transcription reactions using linearized vectors containing T7 or SP6 promoters as templates.

[0067] Transcription was performed using T7 RNA polymerase to synthesize NLS-PmMAD7-NLS mRNA.

[0068] Transcription of NLS-PmMAD7-NLS mRNA includes:

[0069] (a) Sequence characteristics

[0070] * Length: 4252 base pairs

[0071] *Type: Nucleotide

[0072] *Chain type: Single chain

[0073] *Topology: Linear

[0074] 2.5 MAD7-NLS nuclear entry verification

[0075] In order to verify the nuclear entry efficiency of MAD7 nuclease in Penaeus monodon cells after codon optimization, the PmMAD7-NLS-EGFP fusion protein was constructed, and its nuclear entry level was evaluated by microscopic observation of the changes in fluorescence signals after transfection. The results showed that when PmMAD7-NLS mRNA was transfected into Penaeus monodon oocytes, the green fluorescence intensity in the cells was significantly enhanced, indicating that the nuclease successfully entered the cell nucleus and exerted its effect. In contrast, after transfection of PmMAD7-EGFP (without the nuclear localization signal NLS), the observed fluorescence intensity was weak, indicating that the nuclear entry efficiency of PmMAD7 was low when NLS was not added ( Figure 2 ). The present invention significantly improves the efficiency of directional localization in the nucleus of Penaeus monodon cells by introducing a nuclear localization signal (NLS) at the N-terminus and C-terminus of the PmMAD7 nuclease. This optimized design not only enhances the activity of the PmMAD7 nuclease, but also ensures its nuclear entry during the genome editing process.

[0076] 3. crRNA Design and Synthesis

[0077] Gene editing of blood cells of Penaeus monodon

[0078] Before implementing the method of the present invention, first prepare 60-day-old Penaeus monodon, draw blood from it using sodium citrate anticoagulant, prepare blood cells of Penaeus monodon, use L15 basic culture medium, culture at 28° C. in the dark for 24 hours, and then use it for transfection.

[0079] The present invention is further described in detail below in conjunction with specific genes.

[0080] The ECH1 gene is an important gene in the genome of Penaeus monodon, encoding Enoyl-CoA hydratase 1. This enzyme plays a key role in fatty acid metabolism and energy production and is involved in the β-oxidation pathway. Specifically, ECH1 catalyzes the second step of β-oxidation of unsaturated fatty acids, converting 2-enol-CoA into 3-hydroxyacyl-CoA.

[0081] ECH1 is involved in the β-oxidation pathway of fatty acids, a key pathway for lipid breakdown and energy generation. By breaking down fatty acids into acetyl-CoA, ECH1 contributes to the production and maintenance of cellular energy. Through the breakdown of fatty acids and the generation of acetyl-CoA, ECH1 indirectly supports energy production in the tricarboxylic acid cycle (TCA cycle) and the mitochondrial respiratory chain. This is essential for the energy requirements of Penaeus monodon during rapid growth and high metabolic activity. Optimizing ECH1 function may help improve growth efficiency and feed utilization in Penaeus monodon.

[0082] By using the CRISPR-Cas system to perform site-directed mutations or modifications on the ECH1 gene, its specific role in metabolic regulation can be studied. This not only helps to understand the metabolic mechanism of Penaeus monodon, but also provides a molecular basis for genetic improvement of aquaculture performance. Gene editing technology targeting ECH1 may be used to develop shrimp varieties with higher metabolic efficiency and growth rate to improve the economic benefits of aquaculture.

[0083] In order to achieve effective editing of the ECH1 gene of Penaeus monodon, the crRNA of the NLS-PmMAD7-NLS system needs to be precisely designed. The following are the steps for designing the NLS-PmMAD7-NLS target of the ECH1 gene:

[0084] 3.1 Obtaining the ECH1 gene sequence of Penaeus monodon

[0085] First, the complete sequence of the ECH1 gene of Penaeus monodon needs to be obtained, which can be obtained through the public database NCBI, NCBI Reference Sequence: LOC119594195.

[0086] (a) Sequence characteristics

[0087] * Length: 915 base pairs

[0088] *Type: Nucleotide

[0089] *Chain type: double chain

[0090] *Topology: Linear

[0091] (b) Molecule type: Nucleic acid

[0092] Sequence description: LOC119594195.

[0093] 3.2 Target identification and selection:

[0094] Target location: In the ECH1 gene sequence, regions with typical PAM sequences (YTTN) were selected as potential CRISPR targets. Typically, targets located near functional domains or key coding regions are selected to increase the likelihood of phenotypic changes after editing.

[0095] Specificity analysis: Use the online tool Chopchop to perform target specificity analysis. These tools can assess the uniqueness of the selected target in the genome and reduce the risk of off-target editing. Ensure that the selected target has high specificity throughout the genome.

[0096] crRNA design:

[0097] Sequence design: crRNA is designed according to the selected target sequence. crRNA usually includes a 20-nucleotide targeting sequence, adjacent to the PAM sequence.

[0098] Optimization: Optimization of the crRNA sequence may include increasing the GC content to enhance stability and binding efficiency, or introducing modifications into the sequence to improve resistance to nuclease degradation.

[0099] The designed sequence is: gagcaacaucgucaucuccc.

[0100] crRNA synthesis: According to the optimized design, crRNA is synthesized, and chemical modifications need to be introduced when synthesizing mRNA.

[0101] crRNA synthesis specifically includes:

[0102] Chemical modification:

[0103] N1-methyl pseudouridine (m1Ψ) is introduced during the synthesis of crRNA, and chemical modification is performed at the uracil site to replace uridine (U) with N1-methyl pseudouridine. This modification helps reduce the risk of crRNA being degraded by endogenous nucleases in cells and improves its stability and translation efficiency.

[0104] Introduction of N1-methylpseudouridine:

[0105] During in vitro transcription, m1Ψ is introduced using a transcriptase to ensure uniformity and high efficiency of the modification. This modification can reduce the chance of mRNA being recognized by the cellular immune system and reduce the ability to activate pattern recognition receptors such as RIG-I.

[0106] Synthetic sequence:

[0107] Combined with the optimized design, crRNA synthesis was performed, and the sequence after chemical modification was:

[0108] mG*mU*mC*AAAAGACCUUUGGAAUUUCUACUCUUUGUAGAUgagcaacaucgucaucu*mc*mc*mc.

[0109] 4. Gene delivery and verification

[0110] 4.1 Nanocarrier preparation and mRNA embedding

[0111] NLS-PmMAD7-NLS mRNA and crRNA targeting the ECH1 gene were co-encapsulated into MAL-PLGA nanocarriers. During the encapsulation process, the pH and ionic strength of the nanocarriers needed to be precisely controlled to ensure efficient encapsulation rate and carrier stability. The ratio of NLS-PmMAD7-NLS mRNA to crRNA was 1:1, and the total amount was 4 μg.

[0112] 4.2 Preparation of Penaeus monodon hemocytes

[0113] The blood cells of Penaeus monodon prepared in the previous step were pre-cultured: the extracted blood cells were placed in L15 culture medium containing antibiotics and cultured at 28°C for 24 hours to achieve cell recovery and adaptation to the new environment.

[0114] 4.3 Transfection process

[0115] Optimization of transfection conditions: MAL-PLGA nanocarriers containing NLS-PmMAD7-NLS mRNA and crRNA were added to blood cell culture at an mRNA concentration of 200 ng / mL.

[0116] Culture and monitoring: Continue to culture at 28°C for 72 hours, during which time monitor the morphology and activity of the cells to ensure that the cells are not overly stressed.

[0117] 4.4 Genomic DNA extraction and sequencing verification

[0118] DNA extraction: 72 hours after transfection, genomic DNA was extracted from treated blood cells using a commercial DNA extraction kit.

[0119] PCR amplification and sequencing: PCR amplification was performed using primers specific to the ECH1 gene editing region to ensure that only the target region was amplified. Subsequently, the PCR product was sent to high-throughput sequencing for gene editing verification.

[0120] The primer sequences are as follows:

[0121] JC-NGS-ECH1-F:

[0122] TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGagatattggacagtgtttcaacaagc;

[0123] JC-NGS-ECH1-R:

[0124] GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGcgtttctagtgatgaaaatgcatcc.

[0125] Analysis of sequencing results: The sequencing results were compared with the target site region, and overlapping peaks were found in the ECH1 region. After peak resolution and NGS sequencing results, it was shown that the editing site of the ECH1 triple mutant was a deletion mutation ( Figure 3 ).

[0126] 5. Gene editing effect of MAD7 nuclease in Penaeus monodon before and after codon optimization

[0127] In the present study, the gene editing effect of MAD7 nuclease in Penaeus monodon before and after codon optimization was verified by first-generation sequencing. The experimental results showed that ( Figure 4 ), without codon optimization, the target site of the ECH1 gene failed to be successfully edited, and the sequencing results showed a single peak, indicating that the editing failed. After the codon optimization of the mRNA of the MAD7 nuclease, the sequencing results changed significantly, and an obvious double peak appeared at the target site of the ECH1 gene, indicating that successful gene editing occurred at this site. This experimental result further proves that codon optimization significantly improves the translation efficiency of mRNA and enhances the protein expression level, thereby effectively improving the gene editing efficiency of the MAD7 nuclease in Penaeus monodon.

[0128] The above embodiments are only used to illustrate the present invention, and the protection scope of the present invention is not limited to the above embodiments. A person skilled in the art can achieve the purpose of the present invention based on the above disclosure of the present invention, and any improvement and deformation based on the concept of the present invention shall fall within the protection scope of the present invention, and the specific protection scope shall be subject to the claims.

Claims

1. A nucleic acid encoding a fusion protein PmMAD7-NLS for gene editing of Penaeus monodon, characterized in that: The sequence of the nucleic acid encoding the fusion protein PmMAD7-NLS for gene editing of Penaeus monodon is shown in SEQ ID NO:

4.

2. The nucleic acid encoding the fusion protein PmMAD7-NLS for gene editing of Penaeus monodon according to claim 1, characterized in that: The PmMAD7 is a codon-optimized coding sequence of the CRISPR-associated endonuclease MAD7. The nucleotide sequence encoding the CRISPR-associated endonuclease MAD7 is shown in SEQ ID NO: 1, the amino acid sequence of the CRISPR-associated endonuclease MAD7 is shown in SEQ ID NO: 2, and the nucleotide sequence encoding the PmMAD7 is shown in SEQ ID NO:

3.

3. The nucleic acid encoding the fusion protein PmMAD7-NLS for gene editing of Penaeus monodon according to claim 1, characterized in that: The fusion protein PmMAD7-NLS is a codon-optimized CRISPR-associated endonuclease MAD7 modified with a nuclear localization signal NLS. The nuclear localization signal NLS is located at the N-terminus and C-terminus of the codon-optimized CRISPR-associated endonuclease MAD7 protein, and is composed of nuclear localization signals derived from SV40 and nucleoplasmin, respectively.

4. A nucleotide construct for expressing the fusion protein PmMAD7-NLS according to any one of claims 1 to 3, characterized in that: The nucleotide construct includes a nucleotide sequence encoding the fusion protein PmMAD7-NLS.

5. The nucleotide construct according to claim 4, characterized in that Also included are promoters and terminators.

6. The nucleotide construct according to claim 5, characterized in that The promoter is a strong promoter CMV or T7 to ensure high-level expression in Penaeus monodon cells.

7. Use of the fusion protein PmMAD7-NLS according to any one of claims 1 to 3 or the nucleotide construct according to any one of claims 4 to 6 in site-directed editing of the Penaeus monodon genome.

8. The use according to claim 7, characterized in that: The following steps are involved: (1) introducing the nucleotide construct of claim 4 into Penaeus monodon cells; (2) Using the CRISPR-Cas system to induce double-stranded DNA breaks at the target gene site using the fusion protein PmMAD7-NLS; (3) Achieve site-directed mutation, insertion or modification of genes through intracellular DNA repair mechanisms.

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