Anti-myxosporidium gene editing breeding method for carassius auratus var pengze

Abstract

The invention discloses an anti-myxosporidium gene editing breeding method for carassius auratus var pengze, and relates to the technical field of genetic improvement of aquatic animals. According to the invention, light-chemical double controllable split type Cas9 is constructed, and illumination and small molecule induction are performed in a specific time window at the early stage of an embryo, so that a carassius auratus var pengze immune negative regulation gene is synchronously knocked out, and a Tol < 2-> insect-resistant module containing antibacterial peptide is integrated in a targeted manner; and then screening to obtain a stable insect-resistant strain. The method is accurate in editing, low in miss target and short in period, the obtained carassius auratus var pengze can remarkably reduce the myxosporidium infection rate and improve the survival rate, and the method is suitable for large-scale healthy breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic improvement of aquatic animals, and specifically to a gene editing breeding method for Pengze crucian carp resistant to myxosporea. Background Art

[0002] Myxosporidia belong to the class Myxosporida of the phylum Cnidaria and are common parasitic pathogens of freshwater farmed fish. Pengze crucian carp is prone to myxosporidia due to high-density farming, which manifests as tissue vesicles, body deformities and even death, seriously restricting the development of the industry. Existing prevention and control measures include medicated baths, water changes and family selection, but there are still shortcomings such as poor efficacy, high residues and long breeding cycles. In recent years, gene editing tools such as CRISPR / Cas9 have shown potential in aquatic breeding, but traditional systems lack spatiotemporal control, and off-target and ecological risks are still difficult to avoid. Therefore, there is a need for an original gene editing breeding method that is highly precise, safe and controllable and can quickly form a disease-resistant population. Summary of the Invention

[0003] To overcome the deficiencies of the above-mentioned prior art, the present invention provides the following technical solution: constructing a split Cas9 editing enzyme with photo-chemical dual locking, in which the first fragment is fused to the pMag photoreceptor domain, and the second fragment is fused to the nMag photoreceptor domain; the two fragments are further connected to a protein domain that can be drug-induced to dimerize.

[0004] An sgRNA targeting the negative regulatory gene of Pengze crucian carp immunity was designed, as well as an insect-resistant synthetic gene module carrying an anti-microsporidian effector factor, a tissue-specific promoter, and a safety terminator, with Tol2 transposable sequences at both ends of the module.

[0005] A mixed injection solution encoding the above two fragments of Cas9 mRNA, sgRNA and insect-resistant module plasmid was prepared and microinjected into the yolk of one-cell stage Pengze crucian carp fertilized eggs.

[0006] Blue light with a wavelength of 465±10nm is provided in a specific time window in the early embryo, and the corresponding small molecule inducer is added simultaneously to renature Cas9 and perform gene knockout and homologous site-directed insertion; then the light source is turned off and the inducer is removed to inactivate Cas9.

[0007] The F0 generation fry obtained by hatching were screened by fluorescence, PCR and sequencing to confirm the target gene mutation and the integration of the insect-resistant module; negative and off-target individuals were eliminated.

[0008] Positive individuals were selected for artificial insemination to obtain the F1 generation and screen the homozygous edited lines; after the germplasm was established, a myxosporean challenge test was performed to verify its resistance.

[0009] An antimicrobial peptide coding sequence containing a photosensitive non-natural amino acid is further introduced into the anti-insect module, so that the anti-insect activity depends on exogenous feed supplements or specific light, thereby constructing an ecological safety lock.

[0010] The myxosporean-resistant Pengze crucian carp strain obtained by the method showed that the myxosporean load was reduced by ≥90% and the survival rate was increased by ≥50%.

[0011] Compared with the existing technology, the present invention has the following beneficial effects: (1) Gene editing activity is controlled by both light and chemistry, significantly reducing the risk of off-target and sustained expression; (2) The insect-resistant gene module adopts an inducible and tissue-specific expression strategy to achieve on-demand defense and reduce metabolic burden; (3) Through the simultaneous integration and knockout of Tol2-CRISPR, multi-site precise reprogramming is completed within one generation, and the breeding cycle is shortened to 6-9 months; (4) The non-natural amino acid safety lock and triploid-assisted sterility design effectively block the spread of transgenics. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Schematic diagram of the overall process of the method of the present invention. DETAILED DESCRIPTION

[0013] The technical solution of the present invention will be further described below with reference to the accompanying drawings and through specific implementation methods.

[0014] The present invention provides a Pengze crucian carp resistance myxosporean gene editing breeding method, which is characterized by sequentially comprising: S1, constructing a split Cas9 first fragment fused with a light-receptor domain pMag and a drug-dependent dimerization domain X1, and a second fragment fused with nMag and a drug-dependent dimerization domain X2, and obtaining mRNA encoding the first fragment and the second fragment respectively by in vitro transcription; S2, constructing an sgRNA targeting an immune negative regulatory gene of Pengze crucian carp, and preparing an anti-myxosporean synthetic gene module containing a Tol2 sequence; S3, combining step S1 with step S1; The editing enzyme mRNA, sgRNA, insect-resistant module plasmid and Tol2 transposase mRNA obtained in step S2 are mixed and microinjected into one-cell stage Pengze crucian carp fertilized eggs; S4, applying light with a wavelength of 440-490 nm and synchronously adding a dimerization inducer during 0.5-3 hours of embryonic development to renature Cas9 and complete the target gene knockout and targeted integration of the insect-resistant module; S5, removing the light and inducer, hatching the embryos and screening to obtain individuals with both the target gene mutation and the insect-resistant module integration; S6, self-pollinated or backcrossed the individuals obtained in step S5 to establish a myxosporean-resistant strain.

[0015] The photoreceptor domain is pMag / nMag, and the drug-dependent dimerization domain is FRB / FKBP. The anti-insect module includes an epithelial tissue-specific promoter, a target gene encoding an antimicrobial peptide, an inducible or tissue-specific regulatory sequence, and a fluorescent screening marker sequence. The antimicrobial peptide encoding gene contains at least one codon that can encode a photosensitive non-natural amino acid, and the non-natural amino acid is supplemented in the feed. The target gene is NLRC5, SOCS1, SUPPRESSOR OF CYTOKINE SIGNALLING or its homologous gene. The illumination time is 5 to 15 minutes, and the light intensity is 0.5 to 1.2 mWcm -2 The dimerization inducer is selected from a rapamycin analog, a compound ergosterol, or a small molecule with the same function. The method further comprises performing a triploid induction treatment on the fertilized egg before step S3 to make the resulting strain sterile.

[0016] The photochemical (light and drug)-controlled synthetic biology gene editing platform consists of five modules: a gene editing core unit, an optogenetics control unit, a chemical induction control unit, a synthetic biology functional unit, and a delivery and integration unit. These modules work together to achieve precise and controllable modification of the Pengze crucian carp genome and impart resistance to myxosporeans, as follows: The core gene editing unit utilizes a modified CRISPR / Cas system as the editing enzyme. To achieve precise control, the photoactivated Cas9 nuclease (paCas9) is selected as the core tool. paCas9 consists of two segments of the wild-type Cas9 protein, each fused with a photosensitive protein domain, called pMag and nMag. In the absence of light, the two Cas9 segments separate from each other, rendering the enzyme inactive. However, upon exposure to specific wavelengths (such as 465nm blue light), pMag and nMag undergo heterodimerization, reuniting the two Cas9 segments into a functional enzyme, thereby generating a double-strand break at a specific genomic site. This split-and-refold design ensures that gene cleavage occurs only under controlled light signals, significantly enhancing the spatiotemporal controllability of editing. Furthermore, the platform supports the substitution of different Cas enzyme isoforms (such as Cas12a and Cas13) or the use of derivative tools such as base editors and primer editors to meet diverse editing needs.

[0017] Optogenetics control unit: It is composed of an efficient light control system, including an LED light source with adjustable intensity and wavelength, an optical fiber or a microlens array, etc., which is used to precisely irradiate embryos or cells in a culture dish. This unit corresponds to the photosensitive domain of the core Cas9, for example, using blue light to activate pMag / nMag-Cas9. By setting the timing and dosage of light, precise control of the "switch" of editing behavior can be achieved. For example, a brief flash of blue light is used at a specific stage of embryonic development to trigger gene insertion, and then the light source is turned off to inactivate Cas9, thereby avoiding unnecessary continuous cutting. In the future, it can also be expanded to use light control systems with different wavelengths such as red light and far-red light (such as using Phytochrome photoreceptors) to achieve multi-channel independent control of multiple editing events.

[0018] Chemically induced control units: To provide an additional layer of safety and control precision, a small molecule-induced switch is introduced. Specifically, a chemical sensing element is engineered and fused to Cas9 or its guide RNA (gRNA). One approach involves drug-induced split recombination: Cas9 segments are linked to protein domains that require small molecules for dimerization (such as the FKBP / FRB dimerization system). Only upon the addition of a rapamycin analogue, the two Cas9 segments can bind to form an active form. This is similar to the principle of light control, but triggered by a chemical. Another approach involves embedding a drug-activated aptamer switch within the sgRNA. For example, a theophylline-responsive aptamer could be incorporated into the gRNA sequence. In the presence of a specific small molecule, the gRNA secondary structure changes, enabling or disabling Cas9 cleavage. These approaches allow for fine-tuning of editing activity using non-toxic small molecules (such as specific hormones or antibiotic derivatives). Once the desired editing effect is achieved, Cas9 can be deactivated to prevent over-editing or off-target effects.

[0019] Synthetic biology functional units: Simultaneously with genome editing, functional synthetic gene circuits are introduced to confer resistance to myxosporeans in fish. First, through CRISPR-mediated homologous recombination, an insect-resistant gene module is inserted into a safe locus in the Pengze crucian carp (e.g., the MSTN gene, which mediates muscle growth and has no health impact upon deletion). This module can be an antiparasitic factor from another species or a designed gene network. For example, an antimicrosporidian antimicrobial peptide gene (e.g., frog-derived Magainin or black soldier fly-derived antimicrobial peptide) can be inserted to ensure sustained low-level expression of antiparasitic activity in fish tissues, enhancing inherent resistance to infection. Synthetic receptors / immune factors can be introduced (similar to the chimeric antigen receptor concept used in CAR-T) to recognize surface antigens on myxosporean spores and trigger a killing response in fish immune cells. Alternatively, the expression of key endogenous immune regulatory genes can be enhanced, such as by upregulating innate immune recognition receptors like NLRC5, to ensure early and specific recognition of myxosporean spores and initiate immune responses. The expression of these gene modules can be placed under the control of inducible promoters. For example, when a fish experiences an increased inflammatory response to parasite infection, the inducible promoter can drive stronger expression of the insect-resistant gene, thereby providing on-demand enhanced protection when the pathogen is present. Furthermore, to prevent foreign genes from burdening normal physiology, tissue-specific promoters (such as mucosal tissue-specific promoters) can be used to direct the expression of the insect-resistant factor primarily at the parasite's entry point (skin, gill mucosa, etc.), achieving precise defense.

[0020] Delivery and Integration: In actual breeding operations, the above components must be effectively delivered to the nuclei of fertilized eggs or embryos. This delivery is achieved using a modified microinjection-based transposon vector system. First, an mRNA / DNA mixture encoding the two paCas9 fragments, sgRNA, and an insect-resistant gene template is injected into one-cell fertilized eggs via microinjection. To improve integration efficiency, a Tol2 transposon vector, commonly used in fish, is introduced. The insect-resistant gene module is cloned into a plasmid with Tol2 sequences at both ends. Co-injected transposase mRNA mediates efficient insertion into the host genome. Simultaneously, Cas9 performs gene knockout / site-specific insertion editing in early embryos under light / chemical control. By optimizing injection concentration and conditions, the maximum number of embryos is guaranteed to survive and undergo the intended genetic modification. Furthermore, a selectable marker (such as a fluorescent reporter gene) is used to identify individuals with successful integration, facilitating embryonic screening for juveniles that have acquired the insect-resistant module. Positive individuals screened are further verified by PCR and sequencing to confirm the success of the targeted editing (i.e., intended gene knockout and exogenous gene insertion) and to assess for the presence of off-target mutations.

[0021] The five modules, namely the gene editing core unit, optogenetics control unit, chemical induction control unit, synthetic biology functional unit, and delivery and integration unit, constitute an integrated editing platform: using dual switches of light and chemistry to achieve precise control of the editing process, using synthetic biology elements to give the fish new disease resistance functions, and through efficient delivery to ensure that the editing elements act on embryonic cells, so that most individuals can obtain the required genetic modification.

[0022] Optogenetics is being applied to aquaculture genetic breeding, enabling spatiotemporal and temporal selective regulation of gene editing through light-controlled Cas9. This non-invasive photoswitch allows researchers to initiate or terminate gene editing in living fish embryos with subcellular spatial resolution and minute-level temporal precision. This significantly reduces the risk of off-target editing and adverse effects on embryonic development. By integrating dual mechanisms of small molecule and light control, Cas9 remains inactive in the dark and in the absence of inducers, virtually eliminating background activity. Editing is initiated only when both light and chemical induction conditions are met, minimizing the risk of accidental editing or exogenous gene leakage. This multi-level, multi-parameter control far exceeds the safety of existing single-induction systems. The parallelism of spectral and chemical induction enables simultaneous editing of multiple loci in different ways. Two Cas9 variants can be designed to respond to different colors of light (e.g., red light controls another Cas9 to cut a different gene) or different small molecules, allowing them to edit different targets without interfering with each other. In this way, multi-target combination modifications such as "immune negative regulatory gene knockout + insect-resistant gene insertion + growth-related gene optimization" can be completed simultaneously in a single generation.

[0023] A myxosporean-resistant Pengze crucian carp strain obtained by applying a myxosporean-resistant gene editing breeding method for Pengze crucian carp is characterized in that the strain genome contains a Tol2-integrated insect-resistant synthetic gene module and a target gene function loss mutation, and exhibits a cyst reduction rate of ≥90% in a myxosporean infection and toxicity challenge test.

[0024] A gene editing system for a myxospore-resistant gene editing breeding method for Pengze crucian carp is characterized by comprising: mRNAs encoding pMag-Cas9N-X1 and nMag-Cas9C-X2, respectively; sgRNA targeting a target gene; an insect-resistant synthetic gene module plasmid with Tol2 sequences at both ends; Tol2 transposase mRNA; and a small molecule compound for inducing dimerX1 / X2 binding.

[0025] Example 1: Gene editing breeding for resistance to myxosporea: Materials and equipment: 100 pairs of parental Pengze crucian carp; split paCas9 expression plasmid and mRNA in vitro transcription reagent; sgRNA synthesis kit; Tol2 transposase plasmid vector pT2-AMP; LED blue light module (peak 465nm, power 0.8mWcm -2 ); rapamycin analog (AP21967) 30µM final concentration; microinjection system (needle inner diameter 20µm, pressure 40kPa).

[0026] Editing element design: sgRNA selected exon 4 of the negative regulatory region of NLRC5 of Pengze crucian carp, with a target sequence of 5′-GGACTTGCTACCGTGTAGGA-3′; the insect resistance module included: an epithelial tissue-specific promoter (Ep-Pro); a gene encoding the Mag-Opt antimicrobial peptide, with the photosensitive non-natural amino acid Bpa introduced at the key site; an SV40 PolyA signal; Tol2 inverted terminal repeat sequences at both ends; and downstream IRES-mCherry as a screening marker.

[0027] Microinjection and photoinduction / chemical induction: 60 pg each of paCas9-pMag and paCas9-nMag mRNA, 20 pg of sgRNA, 25 pg of Tol2-Mag-Opt plasmid, and 50 pg of Tol2 transposase mRNA were mixed and injected into the yolk of one-cell-stage embryos. Blue light was applied 1.5 hours after fertilization for 10 minutes, and AP21967 was added to the embryo culture medium for 2 hours. The light was then removed, and the medium was changed to remove the inducer.

[0028] Screening and Qualification: 62% of the mCherry positive embryos were detected under fluorescence microscopy at 48 hpf. Genomic PCR analysis revealed the expected indel formation in 18 embryos and integration of the insecticide module in 15. High-throughput sequencing of 10 of these embryos revealed no mutations at the top five predicted off-target sites.

[0029] Evaluation of anti-infection: 30 F0 positive fish and 30 wild type fish were exposed to 2×10 5 Spore ml -1 After 14 days in the experiment, the average number of cysts in the positive group decreased by 93.4% compared with the control, and the survival rate increased by 57.8%.

[0030] Line establishment and genetic stability: F1 lines were obtained by intercrossing F0 positive males and females, and the homozygosity rate of the F1 line was 22% after fluorescence screening. The insect-resistant phenotype remained stable in the F2 generation, conforming to Mendelian segregation.

[0031] Example 2, non-natural amino acid dependent safety lock: Objective: To construct an insect defense line that is activated only under fed-batch conditions by introducing a photosensitive unnatural amino acid p-Benzoyl-L-phenylalanine (pBPA) into the key site of the antimicrobial peptide Mag-Opt and to evaluate the feasibility of a nutrient-dependent safety lock.

[0032] Materials and equipment: pBPA purity ≥99%, Shanghai Yuanye Biotechnology; carp-specific Mj-tRNACUA / Mj-tyrosyl-tRNA synthetase (aaRS) plasmid; 60 pairs of parent Pengze crucian carp; other materials are the same as in Example 1.

[0033] Construction and Injection: A TAG stop codon was placed at position 12 of the amino terminus of Mag-Opt, and the Mj-tRNACUA / aaRS system was used to achieve position-specific encoding of pBPA. Plasmid and mRNA were co-injected into fertilized eggs at the dosages described in Example 1. At 2 hpf, pBPA was administered to the water system at a final concentration of 40 µM for 48 hours.

[0034] Screening and detection: The fluorescence positive ratio was 58%; RT-qPCR showed that the transcription level of anti-insect peptide in the feeding group was 23 times higher than that in the control; LC-MS confirmed that the peptide chain was successfully incorporated into pBPA.

[0035] Functional verification: In the presence of 2×10 myxosporidian spores 5 ml -1 14 days after water poisoning: the cysts of the pBPA-fed group decreased by 91.2%, while those of the non-fed group decreased by only 18.5%, which was not significantly different from the wild type.

[0036] Conclusion: Unnatural amino acids are feasible and can be used for non-proliferation management.

[0037] Example 3, dual-spectrum multi-target collaborative editing: Objective: To construct a blue light-activated Cas9A and a far-red light (650nm)-activated Cas12aB, edit the NLRC5 (blue light) and SOCS1 (red light) genes in parallel, and simultaneously insert a second antimicrobial peptide, Piscidin-1, to achieve multi-target synergistic resistance.

[0038] Materials: Split Cas9A-pMag / nMag mRNA; Split Cas12aB-PhyB / PIF6 mRNA; sgRNA-NLRC5; crRNA-SOCS1; Tol2-Piscidin-1-eGFP vector.

[0039] Methods: After microinjection: 1.5 hpf blue light 465 nm 10 min; 2.0 hpf far-red light 650 nm 8 min.

[0040] Results: The indel rates of NLRC5 and SOCS1 were 74% and 68%, respectively; the double gene mutation rate was 52%, and the Piscidin-1 insertion efficiency was 47%. Fourteen days after challenge, the cyst count decreased by 96.7%, and the survival rate was 85%.

[0041] Example 4, Rapid Breeding of Insect Resistance + Growth Traits: Objective: To knock out the first exon of the MSTN gene while simultaneously knocking out NLRC5 and inserting an insect resistance module using the same platform to increase growth rate.

[0042] Methods: Blue light was used to activate Cas9 to synchronously cut NLRC5 and MSTN; the insect-resistant module was inserted into the "safe harbor" LHFPL5 through homology arms.

[0043] Results: The homozygous rate of NLRC5 and MSTN double knockout was 31%; the average body weight of the F1 generation at 6 months old increased by 35.4%; the myxospore cysts decreased by 94.1%, and there was no significant growth-infection trade-off effect.

[0044] Example 5: 5000m² aquaculture pond pilot test and ecological assessment: Setting: 10,000 conventional seedlings were put into the control pond; 10,000 edited seedlings of Example 3 were put into the test pond; the culture period was 180 days.

[0045] Indicators: Mortality rate in the experimental pond was 4.3%, while that in the control pond was 17.9%; average weight of commercial fish was 476g, while that in the control was 341g; no DNA of escaped edited fish was detected in samples from surrounding waters.

[0046] Conclusion: The edited lines significantly improved yield and health under semi-commercial conditions without causing detectable genetic effects on the environment.

Claims

1. A Pengze crucian carp resistance myxosporea gene editing breeding method, characterized in that: Including in order: S1. Construct a split Cas9 first fragment fused to the photoreceptor domain pMag and the drug-dependent dimerization domain X1, and a second fragment fused to the nMag and the drug-dependent dimerization domain X2. Generate mRNA encoding the first and second fragments, respectively, by in vitro transcription. S2. Construct sgRNA targeting Pengze crucian carp immune negative regulatory genes and prepare anti-myxosporea synthetic gene modules containing the Tol2 sequence; S3, mixing the editing enzyme mRNA, sgRNA, insect-resistant module plasmid, and Tol2 transposase mRNA obtained in step S1 and step S2, and microinjecting the mixture into one-cell-stage Pengze crucian carp fertilized eggs; S4, applying light with a wavelength of 440-490 nm and simultaneously adding a dimerization inducer during 0.5-3 hours of embryonic development to renature Cas9 and complete the target gene knockout and targeted integration of the insect resistance module; S5. Remove light and inducer, hatch the embryos, and screen to obtain individuals that have both the target gene mutation and the insect-resistant module integrated; S6. Self-pollination or backcrossing is performed on the individuals obtained in step S5 to establish a myxosporean-resistant strain.

2. The method for breeding Pengze crucian carp against myxosporea gene editing according to claim 1, characterized in that: The photoreceptor domain is pMag / nMag, and the drug-dependent dimerization domain is FRB / FKBP.

3. The method for breeding Pengze crucian carp against myxosporea gene editing according to claim 2, characterized in that: The anti-insect module includes an epithelial tissue-specific promoter, a target gene encoding an antimicrobial peptide, an inducible or tissue-specific regulatory sequence, and a fluorescent screening marker sequence.

4. The method for breeding Pengze crucian carp against myxosporea gene editing according to claim 3, characterized in that: The antimicrobial peptide encoding gene contains at least one codon encoding a photosensitive non-natural amino acid, and the non-natural amino acid is supplemented in the feed.

5. A Pengze crucian carp resistance to myxosporea gene editing breeding method according to any one of claims 1-4, characterized in that: The target genes are NLRC5, SOCS1, SUPPRESSOR OF CYTOKINE SIGNALLING or their homologous genes.

6. The method for breeding Pengze crucian carp against myxosporea gene editing according to claim 1, characterized in that: The illumination time is 5 to 15 minutes, and the light intensity is 0.5 to 1.2 mW cm -2 .

7. The method for breeding Pengze crucian carp against myxosporea gene editing according to claim 1, characterized in that: The dimerization inducer is selected from rapamycin analogs, compound ergosterol or small molecules with the same function.

8. The method for breeding Pengze crucian carp against myxosporea gene editing according to claim 1, characterized in that: The method further includes performing a triploid induction treatment on the fertilized egg before step S3 to make the obtained strain sterile.

9. A myxospore-resistant Pengze crucian carp strain obtained by applying a gene editing breeding method for Pengze crucian carp resistance is characterized in that: The genome of this strain contains a Tol2-integrated insect-resistant synthetic gene module and a target gene function loss mutation, and in the myxosporean infection and toxicity test, the cyst reduction rate was ≥90%.

10. A gene editing system for the Pengze crucian carp resistance gene editing breeding method, characterized by: Include: mRNA encoding pMag-Cas9N-X1 and nMag-Cas9C-X2, respectively; sgRNA targeting the target gene; An insect-resistant synthetic gene module plasmid with Tol2 sequences at both ends; Tol2 transposase mRNA; Small molecule compounds used to induce dimerX1 / X2 binding.

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