A crisper / dcas9-based zebrafish histone deacetylation editing system

Abstract

The application relates to the technical field of aquaculture, and discloses a CRISPR / dCas9-based zebrafish histone deacetylation editing system, which comprises a core editing module, a target recognition module, an embryo delivery module and a function verification module. Through CRISPR / dCas9 directional fusion and zebrafish akt2 gene specific targeted editing, the target gene histone acetylation level can be accurately regulated, the genomic sequence change is avoided, the akt2 expression can be in-situ down-regulated, and the TOR pathway activity can be inhibited, so that the regulation result is more stable, and a scientific basis is provided for fish energy metabolism related trait improvement.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, and more specifically discloses a zebrafish histone deacetylation editing system based on CRISPR / dCas9. Background Technology

[0002] Aquaculture is a crucial industry for ensuring human food security, and high-quality seedlings constitute a core element of this industry. To improve the traits of economically important aquatic species, practitioners have developed various aquatic breeding techniques. The emergence of epigenetics has provided new insights into breeding; it refers to changes in heritable material within cells other than the DNA sequence, and these changes can be stably continued throughout individual development and generations. This is of great significance for promoting the high-quality development of the current aquatic seed industry.

[0003] In the prior art, document CN106636197B discloses "a method for targeted knockdown of multicopy genes in the zebrafish genome," which includes the following steps: Step 1: preparation of zebrafish embryos; Step 2: construction of expression vector for dCas9Eve fusion protein; Step 3: in vitro transcription of sgRNA, dCas9Eve, Cas9, and the gene, followed by purification of the synthesized sgRNA, dCas9Eve, Cas9 mRNA, and gene mRNA using an RNeasy Mini Kit; Step 4: activity identification of sgRNA; Step 5: knockdown of the expression of the multicopy gene znfl1s using the dCas9Eve method, and determination of gene knockdown status by detecting gene expression using whole embryo in situ hybridization.

[0004] Document CN118147139A discloses "An sgRNA for Targeted Knockout of the SIGIRR Gene in Zebrafish, its CRISPR / Cas9 System, and its Application." This invention provides an sgRNA for knocking out the SIGIRR gene in zebrafish, a primer set for amplifying the sgRNA, and a CRISPR / Cas9 system for knocking out the SIGIRR gene in zebrafish. This invention also provides a method for selecting and knocking out zebrafish with the SIGIRR gene deletion using CRISPR / Cas9 gene editing technology.

[0005] While existing technologies can achieve transcriptional knockdown of multi-copy genes in zebrafish using the dCas9-Eve system to avoid MO toxicity and off-target effects, or obtain gene-deleted zebrafish for bacterial infection research by targeting and knocking out the SIGIRR gene using the CRISPR / Cas9 system, these technologies mostly focus on gene knockdown or DNA-level knockout and have not constructed a fish-specific histone deacetylation editing system. They suffer from problems such as poor element matching, crude targeting design, lack of embryo delivery parameters, and limited detection dimensions. They cannot accurately target the akt2 gene to regulate histone acetylation, nor can they elucidate the relationship between the TOR pathway and glucose metabolism, thus failing to meet the precise regulation needs of aquatic epigenetic breeding. Summary of the Invention

[0006] The main technical problem solved by this invention is to provide a zebrafish histone deacetylation editing system based on CRISPR / dCas9, which can solve the problems mentioned in the background art.

[0007] To solve the above-mentioned technical problems, according to one aspect of the present invention, more specifically, a zebrafish histone deacetylation editing system based on CRISPR / dCas9, comprising: a core editing module, a target recognition module, an embryo delivery module, and a functional verification module; The core editing module constructs a recombinant expression vector with nuclease-free dCas9 and zebrafish hdac1 catalytic domains orientedly fused; The targeting recognition module is designed to target sgRNAs in the upstream and downstream regions of the transcription start site of the zebrafish akt2 gene, and the optimal targeting sgRNA is screened. The embryo delivery module prepares an injection mixture of mRNA transcribed from the recombinant expression vector and optimal sgRNA and delivers it to zebrafish single-cell stage embryos. The functional verification module detects the expression of target genes and pathway-related genes, the level of histone acetylation at target sites, embryonic development and apoptosis, and glucose uptake capacity.

[0008] Furthermore, the core editing module includes: a component selection module, a carrier construction and connection module, and a positioning and fusion module; Component screening module: Screening for SpCas9 mutant dCas9 without nuclease activity, and simultaneously screening for the core functional domain of the zebrafish hdac1 gene containing only histone deacetylation catalytic activity; Vector construction and ligation module: pT3TS-dCas9-MCS plasmid was selected as the vector backbone. The hdac1 catalytic domain fragment and the linearized backbone were ligated directionally after double digestion with NheI and XhoI to construct the pT3TS-dCas9-hdac1CD recombinant expression vector. Localization fusion module: The dCas9 protein C-terminus is fused with the hdac1 catalytic domain in a targeted manner to avoid steric hindrance at the active site after fusion.

[0009] Furthermore, the target recognition module includes: an sgRNA gradient module and an optimal site screening module; sgRNA gradient module: Based on the SpCas9-sgRNA recognition rules, three gradient target sites were designed upstream and downstream of the transcription start site of the akt2 gene, namely 1317 bp upstream, 124 bp upstream, and 458 bp downstream of the start codon. Optimal target selection module: Each sgRNA was combined with mRNA transcribed from the recombinant expression vector and injected into zebrafish embryos. The expression level of the akt2 gene was detected by RT-qPCR, and the sgRNA with the highest editing efficiency was selected as the optimal target sgRNA.

[0010] Furthermore, the embryo delivery module includes: an mRNA preparation and purification module and an embryo microinjection module; mRNA preparation and purification module: After linearizing the recombinant expression vector, dCas9hdac1CD mRNA is synthesized in vitro using the T3 mMESSAGE mMACHINE® Kit, and then purified by RNeasy® Mini Kit to remove untranscribed template and impurities. Embryo microinjection module: AB strain zebrafish single-cell stage embryos were selected, and a mixture containing 400 ng / µL dCas9hdac1CD mRNA and 200 ng / µL optimal sgRNA was precisely injected into the animal pole of the embryo.

[0011] Furthermore, the functional verification module includes: a gene expression detection module, a histone modification detection module, a developmental apoptosis detection module, and a glucose uptake detection module; Gene expression detection module: Total RNA was extracted from embryos using TRNzol reagent, and gapdh was used as an internal reference gene. The expression levels of akt2, TOR pathway, and glucose metabolism-related genes were simultaneously detected by RTqPCR. Histone modification detection module: Using CUT&Tag technology, the Histone H3K9ac and Histone H3K27ac specific antibodies are used to detect the histone acetylation level in the akt2 locus region; Developmental apoptosis detection module: Embryos were cultured at a constant temperature of 28℃. Embryo malformation rate and survival rate were counted at 48 hpf. Embryo cell apoptosis was detected by acridine orange staining at 24 hpf. Glucose uptake detection module: Embryos were incubated in 700 µM 2NBDG working solution in the dark at 72 hpf, and the glucose uptake capacity of the embryos was quantified by fluorescence intensity.

[0012] Furthermore, in the sgRNA gradient module, each gradient sgRNA is a high-purity synthetic sgRNA containing a 20 bp guide sequence and a 3' NGG-type PAM sequence.

[0013] Furthermore, the acridine orange staining concentration was 10 µg / mL, and the incubation time at room temperature in the dark was 30 minutes.

[0014] Furthermore, the hdac1 catalytic domain fragment was amplified using 48 hpf wild-type zebrafish embryo cDNA as a template.

[0015] The beneficial effects of this invention's CRISPR / dCas9-based zebrafish histone deacetylation editing system are as follows: Through CRISPR / dCas9 directed fusion and zebrafish akt2 gene-specific targeted editing, the acetylation level of target genome proteins can be precisely regulated. This not only avoids changes in the genome sequence but also downregulates akt2 expression in situ and inhibits TOR pathway activity, making the regulatory results more stable and providing a scientific basis for improving energy metabolism-related traits in fish. In addition, through embryo-adaptive delivery and multi-dimensional closed-loop verification technology, a balance between editing efficiency and embryo survival rate is achieved, breaking the limitation of poor adaptability of existing epigenetic editing tools and improving the stability and accuracy of editing. At the same time, through nuclease-free epigenetic regulation, off-target risks and ethical controversies are avoided, greatly enhancing the safety and promotional value of the technology. Attached Figure Description

[0016] The present invention will now be described in further detail with reference to the accompanying drawings and specific implementation methods.

[0017] Figure 1 This is a schematic diagram of the system module architecture; Figure 2 This is a schematic diagram of the pT3TS-dCas9-hdac1CD plasmid. Figure 3 This is a schematic diagram showing the location of sgRNA; Figure 4 A schematic diagram illustrating the expression levels of genes related to the akt2 and TOR signaling pathways targeted by the deacetylation editing system; Figure 5 Schematic diagram illustrating the reduction of acetylation levels at the akt2 locus through histone deacetylation editing; Figure 6 A schematic diagram illustrating the targeting of the akt2 embryo phenotype by the deacetylation editing system; Figure 7A schematic diagram of acridine orange staining in embryos targeted by the deacetylation editing system for AKT2; Figure 8 Schematic diagram of 2-NBDG uptake in deacetylated edited embryos; Figure 9 A schematic diagram illustrating the expression levels of genes related to the akt2 glucose transporter targeted by the deacetylation editing system. Detailed Implementation

[0018] The present invention will be described in detail below with reference to the accompanying drawings and embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in the present application can be combined with each other.

[0019] According to one aspect of the invention, such as Figures 1-9 As shown, a CRISPR / dCas9-based zebrafish histone deacetylation editing system is provided, comprising: a core editing module for constructing a recombinant expression vector that directionally fuses a nuclease-free dCas9 with the catalytic functional domain of zebrafish hdac1. This module includes: Component screening module: Screening for SpCas9 mutant dCas9 without nuclease activity, and simultaneously screening for the core functional domain of the zebrafish hdac1 gene containing only histone deacetylation catalytic activity; Specifically, dCas9 with an inactivating mutation was obtained by screening the SpCas9 protein sequence. This mutant completely loses DNA double-strand cutting activity but retains the complete sgRNA guiding target DNA binding ability. For the zebrafish hdac1 gene, its full coding sequence was retrieved from the Ensembl database. The core fragment with only histone deacetylation catalytic activity was identified and extracted using a conserved domain prediction tool. Non-catalytic redundant sequences at the N-terminus and C-terminus were removed to ensure that the subsequent fusion protein has both high catalytic activity and low molecular weight characteristics.

[0020] Vector construction and ligation module: pT3TS-dCas9-MCS plasmid was selected as the vector backbone. The hdac1 catalytic domain fragment and the linearized backbone were ligated directionally after double digestion with NheI and XhoI to construct the pT3TS-dCas9-hdac1CD recombinant expression vector. First, using cDNA from 48 hpf wild-type zebrafish embryos as a template, PCR amplification was performed using a forward primer (F) containing an NheI restriction site and a reverse primer (R) containing an XhoI restriction site. The specific primers are shown in Table 1 below: Table 1

[0021] Obtain the hdac1 catalytic functional domain fragment; Then, the PCR amplification products were purified and recovered using the Tiangen Ultrathin DNA Product Purification Kit. At the same time, the pT3TS-dCas9-MCS plasmid was double-digested with NheI and XhoI restriction endonucleases. After separation by agarose gel electrophoresis, the hdac1 catalytic domain fragment with sticky ends and the linearized vector backbone fragment were recovered respectively. Finally, the recovered target fragment and the vector backbone were added to T4 DNA ligase and the matching buffer at a molar ratio of 3:1, and ligated overnight at 16°C. The ligation product was transformed into DH5α competent cells. After initial screening by colony PCR and verification of complete sequence correctness by Sanger sequencing, the pT3TS-dCas9-hdac1CD recombinant expression vector (e.g., pT3TS-dCas9-hdac1CD) was obtained. Figure 2 (As shown).

[0022] The localization fusion module adopts a method of directional fusion between the C-terminus of the dCas9 protein and the catalytic functional domain of hdac1 to avoid steric hindrance at the active site after fusion; The C-terminus of the dCas9 protein is a flexible, randomly coiled region with flexible spatial conformation. Fusing the hdac1 catalytic domain at this location will not interfere with the specific binding conformation of dCas9 to sgRNA and target DNA. However, if the dCas9 protein is fused with the hdac1 catalytic domain at its N-terminus, the compact N-terminal domain will directly block the catalytic active site of hdac1, resulting in the complete loss of deacetylation function. By using genetic engineering, the coding sequence of the hdac1 catalytic domain is precisely linked to the downstream of the C-terminus of the dCas9 coding sequence to achieve targeted fusion of the two, so that the hdac1 catalytic domain is fully exposed on the surface of the fusion protein, maximizing the histone deacetylation catalytic activity.

[0023] A target recognition module is used to design sgRNAs targeting the upstream and downstream regions of the transcription start site of the zebrafish akt2 gene and to screen for the optimal targeting sgRNAs. This module includes: sgRNA gradient module: Based on the SpCas9-sgRNA recognition rules, three gradient target sites were designed upstream and downstream of the transcription start site of the akt2 gene, namely 1317 bp upstream, 124 bp upstream, and 458 bp downstream of the start codon. First, the complete genome sequence of the zebrafish akt2 gene was retrieved to accurately locate the transcription start site and the upstream and downstream 1500bp regulatory regions. Following the SpCas9-sgRNA recognition rules, potential target sites containing a 20bp specific guide sequence and adjacent to the NGG-type PAM sequence at the 3' end were screened. Genomic repetitive sequences, high GC content regions, and the akt2 gene coding region were avoided throughout the process to ensure high target site specificity and low risk of non-specific binding. Then, three specific target sites with a gradient distribution were precisely selected in the upstream and downstream regions of the akt2 gene transcription start site, corresponding to positions 1317 bp upstream, 124 bp upstream, and 458 bp downstream of the start codon, respectively. These sites cover the distal regulatory region, the proximal promoter core region, and the proximal region after transcription initiation, forming a gradient screening system adapted to different sensitive regions of histone acetylation regulation (such as...). Figure 3 (as shown) Next, the sgRNA sequences corresponding to the three target sites were submitted to synthesize high-purity, chemically unmodified sgRNA. After synthesis, the sgRNA was purified by polyacrylamide gel electrophoresis to avoid interference from impurities in subsequent embryo injection and targeted binding. The preparation and quality control of gradient sgRNA were completed for subsequent targeted editing screening experiments.

[0024] Optimal target selection module: Each sgRNA was combined with mRNA transcribed from the recombinant expression vector and injected into zebrafish embryos. The expression level of the akt2 gene was detected by RT-qPCR, and the sgRNA with the highest editing efficiency was selected as the optimal target sgRNA. Specifically, the pT3TS-dCas9-hdac1CD recombinant plasmid was first linearized by single enzyme digestion, and full-length dCas9-hdac1CD mRNA was synthesized in vitro using the T3mMESSAGE mMACHINE® Kit. Then, the untranscribed template, short RNA fragments and enzyme impurities were removed by purification using the RNeasy® MiniKit. The mRNA was then mixed with three gradient sgRNAs at a concentration ratio of 400 ng / μL mRNA and 200 ng / μL sgRNA to prepare three independent injection mixtures. Then, the three injection mixtures were precisely injected into the animal pole of the single-cell embryo of the AB strain zebrafish using microinjection. Each group was set up with three biological replicates, and each replicate injected no less than 50 healthy embryos. Wild-type embryos that were not injected with any substances were set up as a blank control group to exclude the interference of injection operation and environmental factors on embryonic development and gene expression. Following injection, the embryos were cultured at 28°C until 48 hpf. Thirty embryos were randomly collected from each group. Total RNA was extracted using TRNzol reagent, and cDNA was synthesized using a full-gold reverse transcription kit. AKT2-specific qPCR primers were used, with gapdh as an internal control gene, to quantitatively detect the relative expression level of the AKT2 gene in each group. The primers used are shown in Table 2. Table 2

[0025] Finally, 2 was adopted. -ΔΔCtThe qPCR data were statistically analyzed to compare the downregulation of akt2 gene expression corresponding to the three groups of sgRNAs. The sgRNAs with the lowest akt2 expression level, the most significant downregulation effect, and stable inter-group differences were selected as the optimal target sgRNAs and used as the core target recognition elements for subsequent systemic editing experiments.

[0026] The embryo delivery module prepares an injection mixture of mRNA transcribed from the recombinant expression vector and optimal sgRNA, which is then delivered to zebrafish single-cell stage embryos. This module includes: mRNA preparation and purification module: After linearizing the recombinant expression vector, dCas9hdac1CD mRNA is synthesized in vitro using the T3 mMESSAGE mMACHINE® Kit, and then purified by RNeasy® Mini Kit to remove untranscribed template and impurities. Specifically, the pT3TS-dCas9-hdac1CD recombinant expression vector, which was verified to be correct by sequencing, was used to linearize the circular plasmid by single enzyme digestion with a specific restriction endonuclease. After confirming complete linearization by agarose gel electrophoresis, the linearized plasmid was recovered and purified as an in vitro transcription template. Then, following the standard operating procedure of the T3 mMESSAGE mMACHINE® Kit, the transcription reaction system was prepared. The linearized plasmid template, T3 RNA polymerase, dNTP mixture and other components were mixed in proportion and incubated at 37°C for 2 hours to initiate the in vitro transcription reaction and synthesize complete dCas9-hdac1CD mRNA. Finally, the lysis buffer provided with the kit was added to the transcription reaction solution to terminate the reaction. The solution was then transferred to an RNeasy® MiniKit (QIAGEN, Germany) adsorption column. After binding and multiple washings to remove residual plasmid template, polymerase, and short RNA fragments, high-purity, intact dCas9-hdac1CD mRNA was finally obtained. The mRNA was then aliquoted and stored at -80°C for later use.

[0027] Embryo microinjection module: AB strain zebrafish single-cell stage embryos were selected, and a mixture containing 400 ng / µL dCas9hdac1CD mRNA and 200 ng / µL optimal sgRNA was precisely injected into the animal pole of the embryo. First, healthy, sexually mature zebrafish of the AB strain without genetic defects were selected and placed in a dedicated mating tank at a female-to-male ratio of 1:2. A standard photocycle of 14 hours of light and 10 hours of darkness was set, and natural mating was triggered the next morning. Fertilized eggs were collected. The embryos were rinsed three times with pre-cooled sterile E3 culture medium. Under a stereomicroscope, unfertilized, cloudy, deformed, and embryos that had developed to the 2-cell stage or higher were removed. Healthy embryos with plump morphology, uniform yolk, and in the single-cell stage were selected and transferred to culture dishes containing 1.5% agarose gel for later use. Then, the preserved dCas9-hdac1CD mRNA and optimal sgRNA were taken out, and after brief centrifugation, the injection mixture was precisely prepared with enzyme-free sterile water, so that the final concentration of dCas9-hdac1CD mRNA in the mixture was 400 ng / µL and the final concentration of optimal sgRNA was 200 ng / µL. After gently mixing by pipetting, the mixture was briefly centrifuged for 1 minute to completely remove air bubbles and avoid damaging the embryo during injection. Next, the prepared injection mixture is loaded into a glass injection needle. The parameters are calibrated using a microinjector, and the single injection volume is set to 2 nL to ensure accurate and controllable injection dosage. Finally, under a high-power stereomicroscope, the micromanipulator is manipulated to align the needle tip vertically with the center of the animal pole of the embryo. The needle is gently inserted into the superficial layer of the cytoplasm, triggering the injection device to inject the mixture. The needle is then slowly and steadily withdrawn to avoid cytoplasmic leakage. After injection, the embryos are transferred to fresh, sterile E3 culture medium, with no more than 50 embryos per dish. The dishes are then placed in a 28°C incubator and cultured under 14 hours of light and 10 hours of darkness to complete the entire embryo delivery process.

[0028] The functional validation module detects the expression of target genes and pathway-related genes, histone acetylation levels at target sites, embryonic developmental apoptosis, and glucose uptake capacity. This module includes: Gene expression detection module: Total RNA was extracted from embryos using TRNzol reagent, and gapdh was used as an internal reference gene. The expression levels of akt2, TOR pathway, and glucose metabolism-related genes were simultaneously detected by RTqPCR. Specifically, at 48 hpf, 30 zebrafish embryos were randomly selected from each group, washed twice with pre-cooled PBS to remove surface impurities, and then placed in enzyme-free centrifuge tubes. TRNzol reagent was added to fully lyse the embryonic tissue. After chloroform extraction, isopropanol precipitation, and washing with 75% ethanol, total RNA was obtained, and its concentration and purity were determined. Qualified RNA was used to remove genomic DNA and synthesize cDNA using EasyScript One-Step gDNAR emoval and cDNA Synthesis SuperMix. Using cDNA as a template, amplification was performed on a CFX96 real-time PCR system using PerfectStart® GreenqPCR Super Mix and corresponding specific primers, with three technical replicates. Gapdh was used as an internal control gene. -△△Ct The relative expression levels of genes were calculated using the following method, as shown in Table 3 below: Table 3

[0029] Histone modification detection module: Using CUT&Tag technology, the Histone H3K9ac and Histone H3K27ac specific antibodies are used to detect the histone acetylation level in the akt2 locus region; Specifically, a sufficient number of 48 hpf zebrafish embryos were collected, and a single-cell suspension was prepared by trypsin digestion. After enriching the cells with magnetic beads, Histone H3K9ac or Histone H3K27ac antibodies were added and incubated at room temperature to allow the antibodies to specifically bind to the target histone modification sites. Subsequently, a Protein A / G-Tn5 transposase fusion complex was added, and the cells were recruited to the antibody-binding region by room temperature incubation. 2+ After activation, DNA is cut in situ and inserted into sequencing adapters. Cells are lysed to release DNA fragments, which are then amplified by a small amount of PCR to construct a sequencing library. The library is then subjected to high-throughput sequencing. Bioinformatics analysis is used to compare sequencing peaks in the akt2 locus region and to quantitatively analyze changes in H3K9ac and H3K27ac modification levels (e.g., Figure 5 (As shown).

[0030] Developmental apoptosis detection module: Embryos were cultured at a constant temperature of 28℃. Embryo malformation rate and survival rate were counted at 48 hpf. Embryo cell apoptosis was detected by acridine orange staining at 24 hpf. First, the injected embryos were placed in a 28°C incubator. Fresh E3 culture medium was added every 24 hours, and dead embryos were removed. At 48 hpf, embryo morphology was observed under a stereomicroscope to differentiate between normal, short-tailed, and completely malformed phenotypes. The total number of embryos, the number of malformed embryos, and the number of surviving embryos in each group were counted, and the malformation rate and survival rate were calculated (e.g., ...). Figure 6 (as shown) Then, 24 hpf zebrafish embryos were selected, demeasuring and placed in 24-well plates. They were quickly rinsed three times with E3 culture medium, and acridine orange working solution with a final concentration of 10 µg / mL was added. The plates were then incubated at room temperature in the dark for 30 minutes. Finally, the staining solution was aspirated, and the cells were quickly rinsed three times with E3 culture medium to remove background staining. After Tricaine anesthesia, the cells were placed in methylcellulose, and fluorescence images were captured under the GFP channel. The mean fluorescence intensity of apoptotic cells was quantitatively analyzed using ImageJ software to assess the level of apoptosis (e.g., ...). Figure 7 (As shown).

[0031] Glucose uptake detection module: Embryos were incubated in 700 µM 2NBDG working solution in the dark at 72 hpf, and the glucose uptake capacity of the embryos was quantified by fluorescence intensity. First, 2-NBDG powder was prepared into a 100 nM stock solution using DMSO and stored at -20℃ in the dark. Before the experiment, it was diluted with 1×PBS to prepare a 700 µM working solution. At 72 hpf, 15-20 zebrafish embryos were selected from each group, washed twice with E3 culture medium, and then 1 mL of 2-NBDG working solution was added. The embryos were incubated at 28°C in the dark for 4 hours. After incubation, the embryos were washed multiple times with E3 culture medium to completely remove unbound free 2-NBDG. After Tricaine anesthesia, fluorescence images were captured in the GFP channel, and the overall average fluorescence intensity of the embryos was measured using ImageJ software to quantify differences in glucose uptake capacity (e.g., ...). Figure 8 (As shown).

[0032] To illustrate the deacetylation effect of this invention, we conducted the following experiment: Experimental Objective The effectiveness of the zebrafish histone deacetylation editing system based on CRISPR / dCas9 constructed in this invention in targeting the akt2 gene was verified. Its regulatory effects on the acetylation level of the target gene, TOR pathway activity, embryonic development status and glucose uptake capacity were clarified, and the practicality and specificity of the system in fish epigenetic editing were confirmed.

[0033] Experimental Materials and Methods The experimental subject was AB strain zebrafish; core reagents included pT3TS-dCas9-hdac1CD recombinant plasmid, optimal sgRNA targeting akt2, T3 mMESSAGE mMACHINE® Kit, RNeasy® Mini Kit, TRNzol reagent, EasyScript One-Step gDNA Removal and cDNA Synthesis Super Mix, PerfectStart® Green qPCR Super Mix, Histone H3K9ac antibody, Histone H3K27ac antibody, 2-NBDG, acridine orange, and tricaine; experimental instruments included a stereomicroscope, microinjector, CFX96 real-time PCR system, fluorescence microscope, ImageJ image analysis software, and high-throughput sequencer.

[0034] Experimental process Delivery via editing system: The prepared dCas9-hdac1CD mRNA was mixed with the optimal sgRNA and microinjected into the animal pole of a zebrafish single-cell embryo. Wild-type embryos that were not injected were set as a control group and cultured at a constant temperature of 28°C.

[0035] Histone acetylation detection: The modification levels of H3K9ac and H3K27ac in the akt2 locus region of the edited group and the control group were detected using CUT&Tag technology and specific antibodies.

[0036] Gene expression detection: Embryos were collected at 48 hpf, total RNA was extracted and cDNA was synthesized, and the expression levels of akt2, TOR pathway and glucose metabolism-related genes were detected by RT-qPCR.

[0037] Embryo development and apoptosis detection: Embryo survival rate was recorded during culture, and malformation rate was recorded at 48 hpf; at 24 hpf, acridine orange staining was used to quantitatively analyze cell apoptosis level.

[0038] Glucose uptake assay: Embryos were incubated in 700 µM 2-NBDG working solution in the dark at 72 hpf. The average fluorescence intensity was measured after fluorescence imaging to assess glucose uptake capacity.

[0039] Experimental results Decreased acetylation levels: CUT&Tag analysis showed that the H3K9ac and H3K27ac modification peak signals in the akt2 locus region of the edited group were significantly weaker than those in the control group, confirming that the system successfully achieved target-specific deacetylation.

[0040] Downregulation of target genes and pathway expression: RT-qPCR results showed that akt2 expression was significantly reduced in the edited group, tsc2, a negative regulator of the TOR pathway, was upregulated, and the expression of rheb and core genes of TORC1 (raptor, mlst8, tor) was significantly suppressed, indicating that TOR pathway activity was inhibited (e.g., Figure 4 (As shown).

[0041] Embryonic developmental abnormalities: The embryo malformation rate in the edited group was 51.15%, and the survival rate was significantly lower than that in the control group; 24 hpf acridine orange staining showed that the apoptosis level in the edited group was significantly increased.

[0042] Reduced glucose uptake: 2-NBDG fluorescence detection showed a significant decrease in fluorescence intensity in the edited group, downregulation of glucose transporter-related genes (slc2a1b, slc2a4a, slc2a4b), and a significant decrease in glucose uptake (e.g., Figure 9 (As shown).

[0043] Experimental conclusions The CRISPR / dCas9-hdac1CD histone deacetylation editing system constructed in this invention can precisely target the zebrafish akt2 gene to achieve site-specific deacetylation modification, effectively downregulate the expression of the target gene and inhibit TOR pathway activity, thereby affecting normal embryonic development and glucose metabolism. The system is simple to operate, highly specific, does not change the genomic DNA sequence, and has high safety, providing reliable technical support for the analysis of fish energy metabolism regulation mechanisms and aquatic epigenetic breeding.

[0044] Of course, the above description is not a limitation of the present invention, and the present invention is not limited to the examples given above. Any changes, modifications, additions or substitutions made by those skilled in the art within the scope of the present invention are also within the protection scope of the present invention.

Claims

1. A zebrafish histone deacetylation editing system based on CRISPR / dCas9, characterized in that, include: Core editing module, target recognition module, embryo delivery module, and function verification module; The core editing module constructs a recombinant expression vector with nuclease-free dCas9 and zebrafish hdac1 catalytic domains orientedly fused; The targeting recognition module is designed to target sgRNAs in the upstream and downstream regions of the transcription start site of the zebrafish akt2 gene, and the optimal targeting sgRNA is screened. The embryo delivery module prepares an injection mixture of mRNA transcribed from the recombinant expression vector and optimal sgRNA and delivers it to zebrafish single-cell stage embryos. The functional verification module detects the expression of target genes and pathway-related genes, the level of histone acetylation at target sites, embryonic development and apoptosis, and glucose uptake capacity.

2. The zebrafish histone deacetylation editing system based on CRISPR / dCas9 according to claim 1, characterized in that: The core editing module includes: a component selection module, a carrier construction and connection module, and a positioning and fusion module; Component screening module: Screening for SpCas9 mutant dCas9 without nuclease activity, and simultaneously screening for the core functional domain of the zebrafish hdac1 gene containing only histone deacetylation catalytic activity; Vector construction and ligation module: pT3TS-dCas9-MCS plasmid was selected as the vector backbone. The hdac1 catalytic domain fragment and the linearized backbone were ligated directionally after double digestion with NheI and XhoI to construct the pT3TS-dCas9-hdac1CD recombinant expression vector. Localization fusion module: The dCas9 protein C-terminus is fused with the hdac1 catalytic domain in a targeted manner to avoid steric hindrance at the active site after fusion.

3. The zebrafish histone deacetylation editing system based on CRISPR / dCas9 according to claim 1, characterized in that: The target recognition module includes: an sgRNA gradient module and an optimal site selection module; sgRNA gradient module: Based on the SpCas9-sgRNA recognition rules, three gradient target sites were designed upstream and downstream of the transcription start site of the akt2 gene, namely 1317 bp upstream, 124 bp upstream, and 458 bp downstream of the start codon. Optimal target selection module: Each sgRNA was combined with mRNA transcribed from the recombinant expression vector and injected into zebrafish embryos. The expression level of the akt2 gene was detected by RT-qPCR, and the sgRNA with the highest editing efficiency was selected as the optimal target sgRNA.

4. The zebrafish histone deacetylation editing system based on CRISPR / dCas9 according to claim 1, characterized in that: The embryo delivery module includes: an mRNA preparation and purification module and an embryo microinjection module; mRNA preparation and purification module: After linearizing the recombinant expression vector, dCas9hdac1CD mRNA is synthesized in vitro using the T3 mMESSAGE mMACHINE® Kit, and then purified by RNeasy® Mini Kit to remove untranscribed template and impurities. Embryo microinjection module: AB strain zebrafish single-cell stage embryos were selected, and a mixture containing 400 ng / µL dCas9hdac1CD mRNA and 200 ng / µL of optimal sgRNA was precisely injected into the animal pole of the embryo.

5. The zebrafish histone deacetylation editing system based on CRISPR / dCas9 according to claim 1, characterized in that: The functional verification module includes: a gene expression detection module, a histone modification detection module, a developmental apoptosis detection module, and a glucose uptake detection module; Gene expression detection module: Total RNA was extracted from embryos using TRNzol reagent, and gapdh was used as an internal reference gene. The expression levels of akt2, TOR pathway, and glucose metabolism-related genes were simultaneously detected by RTqPCR. Histone modification detection module: Using CUT&Tag technology, the Histone H3K9ac and Histone H3K27ac specific antibodies are used to detect the histone acetylation level in the akt2 locus region; Developmental apoptosis detection module: Embryos were cultured at a constant temperature of 28℃. Embryo malformation rate and survival rate were counted at 48 hpf. Embryo cell apoptosis was detected by acridine orange staining at 24 hpf. Glucose uptake detection module: Embryos were incubated in 700 µM 2NBDG working solution in the dark at 72 hpf, and the glucose uptake capacity of the embryos was quantified by fluorescence intensity.

6. The zebrafish histone deacetylation editing system based on CRISPR / dCas9 according to claim 3, characterized in that: In the sgRNA gradient module, each gradient sgRNA is a high-purity synthetic sgRNA containing a 20bp guide sequence and a 3' NGG-type PAM sequence.

7. The zebrafish histone deacetylation editing system based on CRISPR / dCas9 according to claim 5, characterized in that: The acridine orange staining concentration was 10 µg / mL, and the incubation time at room temperature in the dark was 30 minutes.

8. The zebrafish histone deacetylation editing system based on CRISPR / dCas9 according to claim 2, characterized in that: The hdac1 catalytic domain fragment was amplified using 48 hpf wild-type zebrafish embryo cDNA as a template.

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