Targeted editing system of methanosarcina hdrE gene, in-vitro evaluation method of influence of methanosarcina hdrE gene on bovine rumen methane generation and application of methanosarcina hdrE gene

Abstract

The invention discloses a target editing system of a methanosarcina hdrE gene, an in-vitro evaluation method of influence of the methanosarcina hdrE gene on bovine rumen methane generation and application, and relates to the technical field of gene editing, and the method comprises the following steps: constructing the target editing system of the methanosarcina hdrE gene; mixing the target editing system of the methane sarcina hdrE gene with the bovine rumen fluid, the silage and the conversion mixed solution to form an in-vitro fermentation system; culturing a fermentation system under the anaerobic condition of 39 DEG C; detecting at least one of the following indexes: methane sarcina relative abundance, methane gas generation amount and rumen microflora structure change; according to the method, the influence of the target editing system of the methanosarcina hdrE gene on bovine rumen methane generation is comprehensively and deeply evaluated from multiple dimensions such as the gene editing effect, the number change of target microorganisms and the whole rumen microorganism ecosystem.

Description

Technical Field

[0001] The present invention relates to the field of gene editing technology, and specifically relates to a targeted editing system for the Methanosarcina barkeri hdrE gene, an in vitro evaluation method for its effect on bovine rumen methane production, and applications thereof. Background Art

[0002] Methane is an important greenhouse gas second only to carbon dioxide, with its global emissions accounting for 16% of the total greenhouse gas emissions, and it has a significant promoting effect on climate warming. In the field of agricultural production, ruminants such as cattle and sheep are the main sources of methane emissions. The microbiota in the rumen of these animals will convert organic matter into substances such as carbon dioxide, volatile fatty acids, and formate during the fermentation of feed. Subsequently, methanogenic archaea in the rumen utilize these substances to finally generate methane gas and emit it into the atmosphere. This process not only causes ruminants to lose 2% - 12% of their feed energy but also significantly exacerbates the greenhouse effect. Since the greenhouse effect of methane is 28 - 36 times that of carbon dioxide, and the global methane emissions from ruminants account for 35% - 40% of the total agricultural emissions, it poses a serious threat to climate change.

[0003] In addition, currently, in the field of evaluating the effect of gene editing technology on the regulation of bovine rumen methane production, there is a lack of a systematic and accurate in vitro evaluation method. Summary of the Invention

[0004] To solve the above technical problems, the present invention aims to provide an in vitro test method based on a targeted editing system for the Methanosarcina barkeri hdrE gene to explore how to reduce bovine methane emissions. This method is economical and effective and does not harm the health of livestock and humans. Specifically as follows: On the one hand, the present invention provides a targeted editing system for the Methanosarcina barkeri hdrE gene, comprising: (a) An sgRNA expression cassette targeting the hdrE gene, whose targeting sequence is as shown in SEQ ID NO:1; hdrE gene sequence: NCBI Gene ID: 1472579; (b) Cas9 protein, whose amino acid sequence: NCBI GenBank accession number is WP_010922251; (c) Ampicillin resistance gene AmpR; (d) pC2A plasmid.

[0005] In the targeted editing system of the Methanosarcina mazei hdrE gene, the sgRNA targets the hdrE gene in Methanosarcina acetivorans. The hdrE gene is located in the genome of Methanosarcina mazei and encodes a subunit (HdrE) of the heterodisulfide reductase (Hdr) complex HdrED. The HdrED complex is a membrane-bound enzyme complex composed of two subunits, HdrE and HdrD, where HdrE is the cytochrome b subunit. The HdrED complex plays a key role in the methanogenesis metabolism of Methanosarcina mazei. It is responsible for reducing the heterodisulfide CoB-S-S-CoM (the disulfide bond between coenzyme B and coenzyme M) to CoB-SH and CoM-SH, and this step is an essential part of the methanogenesis metabolic cycle. CoB-SH and CoM-SH, as electron donors, participate in the final formation of methane under the action of methyl coenzyme M reductase (Mcr). In Methanosarcina mazei, the hdrED operon shows constitutive expression and is essential under all tested growth conditions. The HdrED complex plays a central role in cell survival and methanogenesis metabolism. The deletion of this gene will directly block the formation of the transmembrane proton gradient in methanogens, leading to the collapse of energy metabolism.

[0006] pC2A enables the large-scale replication of this gene editing system in Methanosarcina mazei, and this fragment will not replicate in other archaea and only functions in Methanosarcina mazei, thus avoiding the impact on other rumen microorganisms to a certain extent. Therefore, the targeted editing system of the Methanosarcina mazei hdrE gene is a system that can be expressed in both Methanosarcina mazei and Escherichia coli. This characteristic allows Escherichia coli to be used as the production strain to obtain a large number of expression vectors, while Methanosarcina mazei is used as the recipient strain for modification.

[0007] On the other hand, the present invention provides an in vitro evaluation method for the effect of the targeted editing system of the Methanosarcina mazei hdrE gene on bovine rumen methane production, including the following steps: (1) Construct the targeted editing system of the Methanosarcina mazei hdrE gene; (2) Coat the targeted editing system of the Methanosarcina mazei hdrE gene described in step (1) with PLL-PEG polymer to form PLL-PEG-DNA nanoparticles, and mix them with bovine rumen fluid, silage, and transformation mixture to form an in vitro fermentation system; (3) Cultivate the fermentation system under anaerobic conditions at 39 °C; (4) Detect at least one of the following indicators: the relative abundance of Methanosarcina mazei, the amount of methane gas generated, and the changes in the rumen microbial community structure.

[0008] Further, the sense strand and antisense strand sequences of the sgRNA are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0009] Further, in step (2), each 40 ml fermentation system comprises: 2 ml of PLL-PEG-DNA nanoparticles, 0.5 g of silage, 30 ml of bovine rumen fluid, and 10 ml of transformation mixture.

[0010] Further, the transformation mixture is: 40% PEG4000, 50 mM PIPES buffer (piperazine-1,4-diethanesulfonic acid), 0.35 M sucrose, 0.6 M NaCl, 1 mM MgCl2, 0.00001% resazurin, 0.05% cysteine hydrochloride, and 1 mM DTT (dithiothreitol).

[0011] Further, sodium hydroxide is added dropwise to adjust the pH of the transformation mixture to 7.4.

[0012] Further, in step (3), the incubation time is 24 hours, and the proportion of methane in the generated gas is used as an evaluation index to evaluate the effect.

[0013] On the other hand, the present invention provides an application of a targeted editing system for the hdrE gene of Methanosarcina barkeri, which is used for regulating methane emission from bovine rumen and preparing products for reducing methane production in bovine rumen.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. The in vitro evaluation method for the effect of the targeted editing system for the hdrE gene of Methanosarcina barkeri provided by the present invention can not only detect the methane gas production, but also deeply consider multiple key indicators such as the relative abundance of Methanosarcina barkeri and the changes in the rumen microbial community structure. It can comprehensively and deeply evaluate the effect of the targeted editing system for the hdrE gene of Methanosarcina barkeri on methane production in bovine rumen from multiple dimensions such as the relative abundance of Methanosarcina barkeri, methane gas production, and changes in the rumen microbial community structure, providing a solid basis for accurately judging the role of this gene editing system in reducing bovine methane emissions.

[0015] 2. Traditional in vitro evaluation methods lack effective means for regulating methane emission from bovine rumen by gene editing technology, and the emergence of the present invention fills this gap. Through the technical solution of the present invention, the process of modifying Methanosarcina barkeri can be simulated in vitro, laying a solid foundation for comprehensively and deeply evaluating the effect of the gene editing system on methane production in bovine rumen in the future, making the evaluation work no longer limited to traditional index detection, but exploring from the application level of gene editing technology.

[0016] 3. The present invention first proposes to use the targeted editing system of the Methanosarcina barkeri hdrE gene as a method to reduce methane emissions in cattle. This method allows Escherichia coli to be used as the production strain, while Methanosarcina barkeri is used as the recipient strain. Under the action of the transformation mixture, the gene editing system is delivered into Methanosarcina barkeri, thereby realizing its transformation to reduce the number of Methanosarcina barkeri in the rumen of cattle, and further reducing the methane gas emissions of cattle. The test results show that this method can directly and effectively reduce the methane gas generated during the in vitro fermentation of rumen fluid. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Mass spectrum of Cas9-pC2A-hdrE for Methanosarcina barkeri - Escherichia coli shuttle vector; Figure 2 Agarose gel electrophoresis result diagram containing sgRNA sequence fragments; Figure 3 Agarose gel electrophoresis result diagram of purified linearized DNA plasmid (without sgRNA expression cassette); Figure 4 Total gas production during in vitro fermentation; Figure 5 Proportion of methane gas produced during in vitro fermentation in the total gas production; Figure 6 pH value of the fermentation broth after 24 hours of in vitro fermentation; Figure 7 Change of rumen microbial community in the in vitro fermentation system. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0018] In the following examples, the test materials and reagents used include: ClonExpress II kit (purchased from Novoprotein Scientific Inc.); primers were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0019] In the following examples, the molecular biology test methods not specifically described were all carried out according to the specific methods described in "Molecular Cloning: A Laboratory Manual", or operated according to the kit and product instructions.

[0020] Example 1 Construction of a targeted editing system for the Methanosarcina barkeri hdrE gene 1. A gene editing system capable of replicating and expressing simultaneously in Methanosarcina barkeri and Escherichia coli, namely the targeted editing system of the Methanosarcina barkeri hdrE gene. This system is realized by constructing a Methanosarcina barkeri - Escherichia coli shuttle vector. Figure 1The mass spectrum of Cas9-pC2A-hdrE of the Methanosarcina-Escherichia coli shuttle vector is shown. The mechanism of action is as follows: This system contains a high-copy replication origin, enabling Cas9-pC2A-hdrE to replicate abundantly in Escherichia coli and carry the ampicillin resistance gene. These characteristics allow Escherichia coli to be used as a production strain, and a large amount of Cas9-pC2A-hdrE can be obtained by transforming Escherichia coli. At the same time, pC2A is a natural plasmid fragment present in Methanosarcina, containing multiple genes related to replication in Methanosarcina, thus enabling Cas9-pC2A-hdrE to replicate abundantly in this strain.

[0021] In this system, the sgRNA targets the hdrE gene, which plays a crucial role in the process of methane metabolism. The hdrE gene in Methanosarcina is cleaved by the targeted editing system of the Methanosarcina hdrE gene, ultimately resulting in the breakage of its genome. According to existing research, the genome repair of Methanosarcina mainly relies on the homologous recombination mechanism, and there is no non-homologous recombination. However, it cannot be excluded that there are fragments in Methanosarcina that can support homologous recombination repair. Even if homologous recombination occurs, the mutation or deletion of the hdrE gene will directly block the formation of the transmembrane proton gradient in Methanosarcina, leading to the collapse of energy metabolism and then reducing its quantity. Therefore, by cutting the genome in this way, the number of this archaeon can be effectively reduced, thereby inhibiting methane gas emissions in cattle.

[0022] 2. The sgRNA was selected to target the hdrE gene, and the sgRNA was designed online (the CRISPR-Cas9 sgRNA target design website of the Center for Computational Biology, University of Bergen). The specific sequences are as follows: sgRNA-hdrE-1: Sense strand: SEQ ID NO: 1; Antisense strand: SEQ ID NO: 2.

[0023] 3. To facilitate ligation with the DNA plasmid (without the sgRNA expression cassette), the following primers (Table 1) were designed and entrusted to Sangon Biotech (Shanghai) Co., Ltd. for synthesis. Through PCR amplification, a fragment containing the sgRNA sequence was obtained. After electrophoresis and gel cutting and recovery, the ligation fragment was finally obtained. Figure 2 The agarose gel electrophoresis diagram results of the final ligated fragment (439 bp) are shown; the primer design ensures that the product contains the sgRNA sequence, and there are 15 - 20 bp homologous arms on both sides of the final product to facilitate subsequent homologous recombination.

[0024] Table 1 Primers for constructing the sgRNA expression cassette

[0025] 4. Using the DNA plasmid (without the sgRNA expression cassette) as a template, perform PCR amplification with primers TY-1-F and TY-1-R to obtain fragment 1 containing sgRNA-hdrE-1. At the same time, perform PCR amplification with primers TY-2-F and TY-2-R to obtain fragment 2 containing sgRNA-hdrE-1. After obtaining fragment 1 and fragment 2, use these two fragments as templates and perform PCR amplification with primers TY-1-F and TY-2-R (Tables 2 and 3). After electrophoresis and gel extraction and recovery, the final ligation fragment contains homologous arms of 15-20 bp on each side and the sgRNA sequence.

[0026] Table 2 PCR Program

[0027] Table 3 PCR Reaction System

[0028] 5. To insert the sgRNA expression cassette into the DNA plasmid (without the sgRNA expression cassette), digest the DNA plasmid (without the sgRNA expression cassette) with restriction enzymes SacI and AscI at 37 °C for 1 h, and obtain the linearized fragment through electrophoresis and gel extraction and recovery.

[0029] 6. Figure 3 The agarose gel electrophoresis results of the purified linearized DNA plasmid (without the sgRNA expression cassette) are presented. Carry out the homologous recombination reaction using a homologous recombination kit. The specific operation is as follows: According to the homologous recombination reaction system shown in Table 4, mix the final fragment (i.e., the fragment containing the sgRNA sequence) with the linearized vector, and react at 37 °C for 30 min. After the reaction is completed, transform the above homologous recombination product into DH5α competent cells, spread the transformed cells on an LB plate containing ampicillin, and wait for them to grow. Then, pick single colonies for large-scale culture, and sequence the culture products using the sequencing primer (SEQ ID NO: 7).

[0030] Table 4 Homologous Recombination Reaction System

[0031] 7. The positive clone is cultured and extracted to obtain Cas9-pC2A-hdrE for subsequent experiments. The plasmid endotoxin-free midiprep kit (TIANGEN, China) is used for the extraction of Cas9-pC2A-hdrE.

[0032] Example 2 Bovine In Vitro Fermentation 1. Preparation of Artificial Saliva One day before the experiment, prepare artificial rumen fluid with the ratio of artificial saliva to rumen fluid being 4:1. Prepare 3200 ml of artificial saliva according to the method of Mcdogal. Add 9.80 g of NaHCO3, 0.57 g of KCl, 0.04 g of CaCl2, 9.30 g of NaHPO4•12H2O, 0.47 g of NaCl, 0.12 g of MgSO4·7H2O and 0.25 g of hydrochloride, as well as 0.001 g of resazurin into each liter of water. After mixing the components in proportion, place them in a water bath at 39 °C and heat, while quickly introducing CO2 gas. Seal with plastic wrap to ensure an anaerobic state, stop ventilation when the color changes from blue-violet to transparent and colorless, and then introduce CO2 again before use.

[0033] 2. Collection of rumen fluid Two weeks before the start of the experiment, the experimental animals were fed the total mixed ration (Table 5) provided by the pasture twice a day, at 7:00 am and 17:00 pm respectively. The animals had free access to water and could move around, maintaining regular lighting. The breeding environment was cleaned regularly to minimize errors caused by environmental factors. On the day of collecting rumen fluid, before the morning feeding, select 2 cows with good health conditions, similar parity and body condition, and draw rumen fluid through the rumen cannula connected to a vacuum pump via the oral cavity. After discarding the first 15 ml, collect about 900 ml of rumen fluid. Mix the collected rumen fluid thoroughly, preheat a thermos flask with 39 °C hot water in advance, and fill it with CO2 gas at the same time. Subsequently, filter the rumen fluid through four layers of sterile gauze into the flask and immediately bring it back to the laboratory. Finally, pour the filtered rumen fluid into the artificial saliva and mix well to prepare artificial rumen fluid.

[0034] Table 5 Composition of total mixed ration

[0035] 3. Preparation of PLL-PEG-DNA nanoparticles 3.1. Synthesis of PLL-PEG polymer Materials: Poly-L-lysine hydrobromide (PLL, molecular weight 20 kDa); Methoxypolyethylene glycol-succinimidyl ester (mPEG-NHS, molecular weight 5 kDa); 50 mM sodium tetraborate buffer (pH 8.5); Sterile PBS buffer.

[0036] Steps: 3.1.1. Dissolve 20 mg of PLL in 2 mL of 50 mM sodium tetraborate buffer (pH 8.5) and mix well; 3.1.2. Filter the solution with a 0.22 μm filter membrane to ensure sterility; 3.1.3. Add 25 mg of mPEG-NHS to the filtered PLL solution and stir at room temperature for 6 hours to form a PLL-PEG polymer stock solution.

[0037] 3.2. Assembly of PLL-PEG-DNA nanoparticles Materials: The above-mentioned PLL-PEG polymer stock solution; Cas9-pC2A-hdrE; Sterile PBS buffer.

[0038] Steps: 3.2.1. Dissolve 670 μg of Cas9-pC2A-hdrE in 500 μL of PBS and mix gently.

[0039] 3.2.2. Take 1.38 mL of the PLL-PEG polymer stock solution and mix it with the Cas9-pC2A-hdrE solution, then let it stand at room temperature for 30 minutes to form PLL-PEG-DNA nanoparticles.

[0040] 3.2.3. Make up the total volume to 2 mL with PBS, gently invert and mix, avoiding violent shaking.

[0041] Note: The PLL-PEG polymer is a proportional mixture of poly-L-lysine hydrobromide (PLL) and methoxypolyethylene glycol-succinimidyl ester (mPEG-NHS), which is used to coat the targeted editing system (Cas9-pC2A-hdrE) of the Methanosarcina mazei hdrE gene to form PLL-PEG-DNA nanoparticles.

[0042] 4. Preparation of the transformation mixture The composition of the transformation mixture is as follows: 50 mM PIPES buffer, 0.35 M sucrose, 0.6 M sodium chloride, 1 mM magnesium chloride, 0.00001% resazurin, 0.05% cysteine hydrochloride, 1 mM DTT, 40% polyethylene glycol 4000, and the pH value of the transformation mixture is 7.4.

[0043] 5. Experimental design The fermented substrate was selected as natural forage silage, and its source was consistent with the diet of the rumen fluid donor animals. After being dried at 65 °C, the substrate was crushed and passed through a 45-mesh sieve. A total of three groups were set up in the experiment (Table 6): the blank group, the control group, and the experimental group. Each group had 6 replicates, and the total volume of each sample was 40 ml. The whole experiment was carried out in a water bath to simulate the environment of 39 °C in the rumen. In a culture bottle containing 0.5 g of fermented substrate, the corresponding fermentation system was configured. After configuration, CO2 was immediately introduced for 3 - 5 seconds to displace the air. Subsequently, it was sealed with a rubber stopper, and a 60-ml syringe was connected to the rubber stopper through a two-way valve. Then all interfaces were wrapped with tape to prevent air leakage. The culture bottle was placed in a constant temperature water bath for in vitro fermentation culture. After fermentation, the fermentation broth was filtered and stored at -80 °C for later use.

[0044] Table 6 Experimental group system

[0045] Note: In the PEG group, the DNA was Cas9-pC2A-hdrE.

[0046] 6. Methane gas analysis The gas components collected by the syringe were determined by gas chromatography to analyze the percentage concentration of methane during in vitro fermentation. The gas production of each component was calculated by the following formula: Gas production of each component = Total gas production in 24 hours × Percentage of this component. The total gas production in 24 hours was counted through scale readings. Nitrogen was used as the carrier gas in the experiment. The gas flow rate was set at 20 ml / min, the injection volume was 1 ml, the column oven temperature was 80 °C, the thermal conductivity cell temperature was set at 150 °C, and the external standard method was used for calculation.

[0047] 7. Determination of pH value of fermentation broth After calibrating the pH meter (PB-10 Sartorius) with reference to the GB / T41439-2022 standard, the pH value of the filtrate was measured. Under repeatability conditions, the absolute difference between the two independent measurement results did not exceed 0.2.

[0048] 8. Data processing GraphPad Prism 10.1.2 was used for graph drawing, and SPSS 26.0 software was used to analyze the experimental data. P < 0.05 was considered a significant difference, P < 0.01 was considered a highly significant difference, and P < 0.001 was considered an extremely significant difference.

[0049] 9. Results The results are shown in Figures 4 - 6 and Table 7. Figure 4 was the total gas production during in vitro fermentation, Figure 5 was the proportion of methane gas produced during in vitro fermentation in the total gas production, Figure 6is the pH value of the fermentation broth after 24 hours of in vitro fermentation; in the figure, * indicates a significant difference between the two groups, ** indicates a highly significant difference between the two groups, and *** indicates an extremely significant difference between the two groups.

[0050] Table 7 Effects of different treatments on methane production and pH value

[0051] From Figures 4 - 6 and Table 7, it can be seen that after introducing the targeted editing system of the Methanosarcina hdrE gene into the conversion mixture in the experimental group, the proportion of methane gas in the total gas decreased significantly, while the overall gas production remained relatively stable. This indicates that the method used in the present invention has an obvious effect in reducing methane emissions.

[0052] In addition, the research results show that the pH value of the rumen fluid after treatment showed a certain degree of change. It is worth noting that this change still remains within the pH range of normal rumen fermentation in cattle. Combining the results of rumen microorganism qPCR analysis, it can be seen that the method adopted in the present invention effectively reduces the relative abundance of methanogens, thereby adjusting the microbial community structure in the in vitro fermentation system. From a macroscopic perspective, this adjustment has an impact on the acid-base balance of the fermentation environment, but does not damage the normal physiological functions of the rumen, thus ensuring the overall stability of the rumen fermentation process.

[0053] Example 3 Analysis of rumen microorganisms in in vitro fermentation Use the E.Z.N.A.® Soil DNA Kit kit and follow its instructions to extract DNA from the preserved fermentation broth. Evaluate the concentration and purity of the extracted genomic DNA by measuring the A260 / 280 ratio, and verify it using 1% agarose gel. Dilute the DNA sample to 10 ng / μL, and then use qPCR to quantitatively analyze the relative abundance of rumen microorganisms. The measured microorganisms include Bifidobacterium, Methanogens, Ruminococcus albus, Lactobacillus, Bacillus, and Actinobacillus succinogenes, and calculate their relative abundances.

[0054] The qPCR amplification reaction was carried out in a 20 μL system, and the reaction system included 10 μL SYBR Premix Taq™ (TaKaRa Biotechnology Co., Ltd., Dalian, China), 1.5 μL primers, 5 μL ddH2O, and 2 μL (10 ng / μL) DNA template. The amplification program was as follows: pre-denaturation at 95 °C for 1 min, followed by 40 cycles (95 °C for 30 s, annealing at 60 °C for 30 s, and extension at 68 °C for 1 min). The cycle threshold (Ct value) was recorded during the amplification process and used to calculate the relative abundance of each target microorganism relative to the total rumen bacteria. The formula for calculating the relative abundance of microorganisms is as follows: Relative abundance = 2 -[ Ct（target）－Ct (total bacteria)] , where Ct target is the Ct value measured by the primers of the target bacteria, and Ct total bacteria is the Ct value obtained with the primers of the total bacteria.

[0055] The specific primer sequences used for qPCR are shown in Table 8.

[0056] Table 8 Specific primer sequences for qPCR

[0057] The effects of different treatment groups on microorganisms in the in vitro fermentation system are shown in Table 9 and Figure 7 as follows. Figure 7 For the comparison of the content of each microorganism with the total bacteria as a reference, the specific microorganisms measured were Bifidobacterium (Bifidobacterium ×10 -3 ), Methanogens (Methanogens ×10 -3 ), Ruminococcus albus (Ruminococcus albus ×10 -4 ), Lactobacillus (Lactobacillus ×10 -1 ), Bacillus (Bacillus ×10⁻³), and Actinobacillus succinogenes (Actinobacillus succinogenes ×10⁻²).

[0058] Table 9 Effects of different treatment groups on microorganisms in the in vitro fermentation system

[0059] Compared with the control group and the blank group, during the in vitro rumen fermentation process of the experimental group, the abundance of specific microorganisms changed significantly. Specifically, when the targeted editing system of the hdrE gene of Methanosarcina was introduced into the conversion mixture, the abundance of methanogens decreased significantly (P≤0.001). This change indicates that the method of the present invention can effectively inhibit the growth of methanogens, thereby reducing methane production and achieving the goal of reducing methane emissions.

[0060] At the same time, significant changes in the abundance of other microorganisms were also observed. Among them, Ruminococcus albus, Bacillus, and Lactobacillus all showed fluctuations in abundance. This phenomenon may be due to the influence of different conversion systems on the microbial community in the original rumen fluid, and this change is manifested as a change in the pH value of the fermentation broth at the macroscopic level.

Claims

1. A targeted editing system for the hdrE gene of Methanosarcina, characterized in that: The targeted editing system comprises: (a) an sgRNA expression cassette targeting the hdrE gene, wherein the targeting sequence of the sgRNA expression cassette is shown in SEQ ID NO: 1; (b) Cas9 protein; (c) ampicillin resistance gene AmpR; (d) pC2A plasmid.

2. An in vitro evaluation method for the effect of the targeted editing system of the hdrE gene of Methanosarcina as claimed in claim 1 on bovine rumen methane production, characterized in that: The following steps are involved: (1) Construction of a targeted editing system for the hdrE gene of Methanosarcina; (2) using a PLL-PEG polymer to encapsulate the targeted editing system of the hdrE gene of Methanosarcina described in step (1) into PLL-PEG-DNA nanoparticles, and mixing them with bovine rumen fluid, silage, and conversion mixture to form an in vitro fermentation system; (3) Cultivating the fermentation system under anaerobic conditions at 39°C; (4) Detect at least one of the following indicators: relative abundance of Methanosarcina, methane gas production, and changes in the structure of rumen microbial community.

3. The in vitro evaluation method according to claim 2, characterized in that In step (2), each 40 ml fermentation system contains: 2 ml PLL-PEG-DNA nanoparticles, 0.5 g silage, 30 ml bovine rumen fluid and 10 ml conversion mixture.

4. The in vitro evaluation method according to claim 2, characterized in that The transformation mixture is composed of: 40% PEG4000, 50 mM PIPES buffer, 0.35 M sucrose, 0.6 M NaCl, 1 mM MgCl2, 0.00001% resazurin, 0.05% cysteine ​​hydrochloride and 1 mM DTT.

5. The in vitro evaluation method according to claim 3, characterized in that: Sodium hydroxide was added dropwise to adjust the pH of the conversion mixture to 7.

4.

6. The in vitro evaluation method according to claim 2, characterized in that: In the step (3), the culture time is 24 hours, and the effect is evaluated by taking the proportion of methane in the generated gas as an evaluation index.

7. Use of a targeted editing system for the hdrE gene of Methanosarcina as claimed in claim 1, characterized in that: Used to regulate cattle rumen methane emissions.

8. Use of a targeted editing system for the hdrE gene of Methanosarcina as claimed in claim 1, characterized in that: Used to prepare products that reduce methane production in the rumen of cattle.

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