A targeted editing system for the hdrE gene of Methanosarcina and its in vitro evaluation method and application for its effect on bovine rumen methane production

Abstract

The present invention discloses a targeted editing system of the hdrE gene of Methanosarcina and an in vitro evaluation method and application of its effect on bovine rumen methane production, relating to the field of gene editing technology, comprising the following steps: constructing a targeted editing system of the hdrE gene of Methanosarcina; mixing the targeted editing system of the hdrE gene of Methanosarcina with bovine rumen fluid, silage, and conversion mixture to form an in vitro fermentation system; culturing the fermentation system under anaerobic conditions at 39°C; and detecting at least one of the following indicators: relative abundance of Methanosarcina, methane gas production, and changes in rumen microbial community structure. The present invention comprehensively and deeply evaluates the effect of the targeted editing system of the hdrE gene of Methanosarcina on bovine rumen methane production from multiple dimensions, including gene editing effect, changes in the number of target microorganisms, and the entire rumen microbial ecosystem.

Description

Technical Field

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

[0002] Methane is the second most important greenhouse gas after carbon dioxide, accounting for 16% of total global greenhouse gas emissions and significantly contributing to climate warming. In agricultural production, ruminants such as cattle and sheep are the main sources of methane emissions. During the fermentation of feed, the microbial communities in the rumen of these animals convert organic matter into substances such as carbon dioxide, volatile fatty acids, and formate. Subsequently, methanogenic archaea in the rumen utilize these substances to ultimately generate methane gas, which is released into the atmosphere. This process not only causes ruminants to lose 2%-12% of their feed energy, but also significantly exacerbates the greenhouse effect. Because methane's greenhouse effect is 28-36 times that of carbon dioxide, and global ruminant methane emissions account for 35%-40% of total agricultural emissions, it poses a serious threat to climate change.

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

[0004] To address the above technical problems, the present invention aims to provide an in vitro experimental method based on a targeted editing system of the hdrE gene of Methanosarcina to explore how to reduce methane emissions from cattle. This method is economical and effective, and does not cause harm to livestock and human health. The details are as follows:

[0005] In one aspect, the present invention provides a targeted editing system for the hdrE gene of Methanosarcina, comprising:

[0006] (a) sgRNA expression cassette targeting the hdrE gene, whose targeting sequence is shown in SEQ ID NO: 1; hdrE gene sequence: NCBI Gene ID: 1472579;

[0007] (b) Cas9 protein, amino acid sequence: NCBI GenBank accession number: WP_010922251;

[0008] (c) ampicillin resistance gene AmpR;

[0009] (d) pC2A plasmid.

[0010] In the targeted editing system for the hdrE gene of Methanosarcina, sgRNA targets the hdrE gene in Methanosarcina acetica. The hdrE gene, located in the genome of Methanosarcina, encodes a subunit (HdrE) of the heterodisulfide reductase (Hdr) complex. The HdrED complex is a membrane-bound enzyme composed of two subunits, HdrE and HdrD, with HdrE being a cytochrome b subunit. The HdrED complex plays a key role in Methanosarcina's methanogenic metabolism, responsible for reducing the heterodisulfide CoB-SS-CoM (the disulfide bond between coenzyme B and coenzyme M) to CoB-SH and CoM-SH, a necessary step in the methanogenic metabolic cycle. CoB-SH and CoM-SH act as electron donors, contributing to the final production of methane through the action of methyl-CoA reductase (Mcr). In Methanosarcina, the hdrED operon is constitutively expressed and essential under all growth conditions tested. The HdrED complex plays a central role in cell survival and methanogenic metabolism. Deletion of this gene directly blocks the formation of a transmembrane proton gradient in methanogens, leading to a collapse of energy metabolism.

[0011] pC2A enables the gene editing system to replicate in large quantities in Methanosarcina, while the fragment will not replicate in other archaea and will only work in Methanosarcina, thereby avoiding the impact on other rumen microorganisms to a certain extent. Therefore, the targeted editing system of the Methanosarcina hdrE gene is a system that can be expressed in both Methanosarcina and Escherichia coli. This feature makes it possible to use Escherichia coli as a production strain to obtain a large amount of expression vectors, while Methanosarcina is used as a recipient strain for transformation.

[0012] In another aspect, the present invention provides an in vitro method for evaluating the effect of a targeted editing system of the hdrE gene of Methanosarcina on bovine rumen methane production, comprising the following steps:

[0013] (1) Construction of a targeted editing system for the hdrE gene of Methanosarcina;

[0014] (2) The targeted editing system of the Methanosarcina hdrE gene described in step (1) is coated into PLL-PEG-DNA nanoparticles using a PLL-PEG polymer, and the nanoparticles are mixed with bovine rumen fluid, silage, and a conversion mixture to form an in vitro fermentation system;

[0015] (3) Cultivate the fermentation system under anaerobic conditions at 39°C;

[0016] (4) Detect at least one of the following indicators: relative abundance of Methanosarcina, methane gas production, and changes in the structure of rumen microbial communities.

[0017] Furthermore, the sense strand and antisense strand sequences of the sgRNA are shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0018] Furthermore, in step (2), each 40 ml fermentation system comprises: 2 ml PLL-PEG-DNA nanoparticles, 0.5 g silage, 30 ml bovine rumen fluid and 10 ml conversion mixture.

[0019] Furthermore, the transformation mixture is composed of: 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).

[0020] Further, sodium hydroxide was added dropwise to adjust the pH of the conversion mixture to 7.4.

[0021] Furthermore, in step (3), the culture time is 24 hours, and the effect is evaluated using the proportion of methane in the generated gas as an evaluation index.

[0022] In another aspect, the present invention provides an application of a targeted editing system for the hdrE gene of Methanosarcina for regulating bovine rumen methane emissions and preparing products that reduce bovine rumen methane production.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The present invention provides an in vitro evaluation method for the effect of a targeted editing system of the hdrE gene of Methanosarcina on methane production in the rumen of cattle. The method can not only detect the amount of methane gas produced, but also deeply consider multiple key indicators such as the relative abundance of Methanosarcina and changes in the structure of rumen microbial communities. It can comprehensively and deeply evaluate the effect of the targeted editing system of the hdrE gene of Methanosarcina on methane production in the rumen of cattle from multiple dimensions such as the relative abundance of Methanosarcina, methane gas production, and changes in the structure of rumen microbial communities, providing a solid basis for accurately judging the role of the gene editing system in reducing methane emissions from cattle.

[0025] 2. Traditional in vitro evaluation methods lack effective means to regulate bovine rumen methane emissions through gene editing technology, and the emergence of the present invention fills this gap. Through the technical solution of the present invention, the transformation process of Methanosarcina can be simulated in vitro, laying a solid foundation for the subsequent comprehensive and in-depth evaluation of the impact of the gene editing system on bovine rumen methane production. The evaluation work is no longer limited to traditional indicator detection, but a new exploration is carried out from the application level of gene editing technology.

[0026] 3. The present invention proposes for the first time the use of a targeted editing system of the hdrE gene of Methanosarcina as a method for reducing methane emissions from cattle. This method allows Escherichia coli to be used as a production strain and Methanosarcina to be used as a recipient strain. Under the action of a transformation mixture, the gene editing system is delivered to Methanosarcina, thereby achieving its transformation to reduce the number of Methanosarcina in the rumen of cattle, thereby reducing methane gas emissions from cattle. Experimental results show that this method can directly and effectively reduce the methane gas produced during the fermentation process outside the rumen liquid. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the mass spectrum of Cas9-pC2A-hdrE of the Methanosarcina-Escherichia coli shuttle vector;

[0028] Figure 2 This is the result of agarose gel electrophoresis of the sgRNA sequence fragment;

[0029] Figure 3 The figure shows the agarose gel electrophoresis results of the purified linearized DNA plasmid (without the sgRNA expression cassette);

[0030] Figure 4 is the total gas production during in vitro fermentation;

[0031] Figure 5 is the ratio of methane gas produced during in vitro fermentation to the total gas production;

[0032] Figure 6 is the pH value of the fermentation liquid after 24 h of in vitro fermentation;

[0033] Figure 7 The figure shows the changes of rumen microbial community in the in vitro fermentation system. DETAILED DESCRIPTION

[0034] In the following examples, the experimental materials and reagents used include: ClonExpress II kit (purchased from Novozymes Biotech Co., Ltd.); primers synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0035] In the following examples, molecular biology experimental methods not specifically described were performed according to the specific methods described in the Molecular Cloning Experiment Guide, or according to the kits and product instructions.

[0036] Example 1 Construction of a targeted editing system for the hdrE gene of Methanosarcina

[0037] 1. A gene editing system capable of simultaneous replication and expression in Methanosarcina and Escherichia coli, namely a targeted editing system for the hdrE gene of Methanosarcina, which is achieved by constructing a Methanosarcina-Escherichia coli shuttle vector. Figure 1 The mass spectrum of Cas9-pC2A-hdrE from 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 extensively in E. coli, and carries an ampicillin resistance gene. These properties allow E. coli to be used as a production strain, allowing large quantities of Cas9-pC2A-hdrE to be obtained through transformation. Furthermore, pC2A is a natural plasmid fragment present in Methanosarcina and contains multiple genes involved in replication in Methanosarcina, enabling Cas9-pC2A-hdrE to replicate extensively in this strain.

[0038] In this system, sgRNA targets the hdrE gene, which plays a vital role in methane metabolism. The hdrE gene in Methanosarcina is cut by the targeted editing system of the hdrE gene of Methanosarcina, which eventually leads to 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 is not ruled out 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 still directly block the formation of a transmembrane proton gradient in Methanosarcina, thereby causing the collapse of energy metabolism and reducing its number. Therefore, by cutting off the genome in this way, the number of this archaeon can be effectively reduced, thereby inhibiting methane gas emissions from cattle.

[0039] 2. sgRNA selection targeting the hdrE gene. sgRNA was designed online (CRISPR-Cas9 sgRNA target design website of the Center for Computational Biology, University of Bergen). The specific sequence is as follows:

[0040] sgRNA-hdrE-1: positive strand: SEQ ID NO: 1;

[0041] Antisense strand: SEQ ID NO: 2.

[0042] 3. To facilitate ligation with a DNA plasmid (without the sgRNA expression cassette), the following primers were designed (Table 1) and synthesized by Sangon Biotech (Shanghai) Co., Ltd. PCR amplification yielded a fragment containing the sgRNA sequence. After electrophoresis and gel extraction, the ligated fragment was obtained. Figure 2 The agarose gel electrophoresis results of the final ligated fragment (439 bp) are shown; the primer design ensures that the product contains the sgRNA sequence and has 15-20 bp homology arms on both sides of the final product to facilitate subsequent homologous recombination.

[0043] Table 1 Primers for constructing sgRNA expression cassettes

[0044]

[0045] 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 the sgRNA-hdrE-1. Simultaneously, perform PCR amplification with primers TY-2-F and TY-2-R to obtain fragment 2 containing the sgRNA-hdrE-1. After obtaining fragments 1 and 2, perform PCR amplification using primers TY-1-F and TY-2-R (Tables 2 and 3) using these fragments as templates. After electrophoresis and gel extraction, the resulting ligated fragment contains 15-20 bp of homology arms on each side and the sgRNA sequence.

[0046] Table 2 PCR program

[0047]

[0048] Table 3 PCR reaction system

[0049]

[0050] 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 endonucleases SacI and AscI at 37°C for 1 hour. Recover the linearized fragment by electrophoresis and gel excision.

[0051] 6. Figure 3Agarose gel electrophoresis results of the purified linearized DNA plasmid (without the sgRNA expression cassette) are presented. Homologous recombination reactions were performed using a homologous recombination kit. Specifically, according to the homologous recombination reaction system shown in Table 4, the final fragment (i.e., the fragment containing the sgRNA sequence) was mixed with the linearized vector and reacted at 37°C for 30 minutes. After completion of the reaction, the homologous recombination product was transformed into DH5α competent cells, plated on LB plates containing ampicillin, and allowed to grow. Subsequently, single colonies were picked for expansion and sequenced using a sequencing primer (SEQ ID NO: 7).

[0052] Table 4 Homologous recombination reaction system

[0053]

[0054] 7. Positive clones were cultured and extracted to obtain Cas9-pC2A-hdrE for subsequent experiments. Cas9-pC2A-hdrE was extracted using a plasmid endotoxin removal kit (TIANGEN, China).

[0055] Example 2 In vitro fermentation of cattle

[0056] 1. Preparation of artificial saliva

[0057] The day before the experiment, prepare artificial rumen fluid (ARF) at a ratio of 4:1. Prepare 3200 ml of ARF according to the McDogel method. Add 9.80 g NaHCO₃, 0.57 g KCl, 0.04 g CaCl₂, 9.30 g NaHPO₄·12H₂O, 0.47 g NaCl, 0.12 g MgSO₄·7H₂O, 0.25 g hydrochloride, and 0.001 g resazurin per liter of water. Mix the ingredients in the appropriate proportions and heat in a 39°C water bath while rapidly bubbling with CO₂. Seal the container with plastic wrap to ensure anaerobic conditions. Stop bubbling when the color changes from bluish-purple to transparent and colorless. Re-bubble with CO₂ just before use.

[0058] 2. Collection of rumen fluid

[0059] For two weeks prior to the start of the experiment, experimental animals were fed a total mixed diet provided by the farm (Table 5) twice daily, at 7:00 AM and 5:00 PM. Animals had free access to water and exercise, and regular lighting was maintained. The housing environment was cleaned regularly to minimize environmental variations. On the day of rumen fluid collection, before morning feeding, two cows in good health, of similar parity and body condition were selected. Rumen fluid was extracted orally using a rumen cannula connected to a vacuum pump. After discarding the first 15 ml, approximately 900 ml of rumen fluid was collected. The collected rumen fluid was thoroughly mixed and placed in a thermos preheated with 39°C hot water and filled with CO2. The rumen fluid was then filtered through four layers of sterile gauze into a bottle and immediately brought back to the laboratory. Finally, the filtered rumen fluid was poured into artificial saliva and mixed to prepare artificial rumen fluid.

[0060] Table 5 Total mixed ration composition

[0061]

[0062] 3. Preparation of PLL-PEG-DNA Nanoparticles

[0063] 3.1 Synthesis of PLL-PEG Polymer

[0064] Material:

[0065] poly-L-lysine hydrobromide (PLL, molecular weight 20 kDa);

[0066] methoxypolyethylene glycol-succinimidyl ester (mPEG-NHS, molecular weight 5 kDa);

[0067] 50 mM sodium tetraborate buffer (pH 8.5);

[0068] Sterile PBS buffer.

[0069] step:

[0070] 3.1.1. Dissolve 20 mg of PLL in 2 mL of 50 mM sodium tetraborate buffer (pH 8.5) and mix thoroughly.

[0071] 3.1.2. Filter the solution through a 0.22 μm filter membrane to ensure sterility.

[0072] 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.

[0073] 3.2. Assembly of PLL-PEG-DNA Nanoparticles

[0074] Material:

[0075] The above-mentioned PLL-PEG polymer stock solution;

[0076] Cas9-pC2A-hdrE;

[0077] Sterile PBS buffer.

[0078] step:

[0079] 3.2.1. Dissolve 670 μg of Cas9-pC2A-hdrE in 500 μL of PBS and mix gently.

[0080] 3.2.2. Take 1.38 mL of the PLL-PEG polymer stock solution, mix it with the Cas9-pC2A-hdrE solution, and let it stand at room temperature for 30 minutes to form PLL-PEG-DNA nanoparticles.

[0081] 3.2.3. Add PBS to make up the total volume to 2 mL and mix gently by inversion. Avoid vigorous shaking.

[0082] Note: PLL-PEG polymer is a mixture of poly-L-lysine hydrobromide (PLL) and methoxypolyethylene glycol-succinimidyl ester (mPEG-NHS) in equal proportions and is used to coat the targeted editing system (Cas9-pC2A-hdrE) of the Methanosarcina hdrE gene to form PLL-PEG-DNA nanoparticles.

[0083] 4. Preparation of transformation mixture

[0084] 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, and 40% polyethylene glycol 4000. The pH value of the transformation mixture is 7.4.

[0085] 5. Experimental Design

[0086] The fermentation substrate was natural grass silage, sourced from the same diet as the rumen fluid donor animals. After drying at 65°C, the substrate was ground and passed through a 45-mesh sieve. Three groups were included (Table 6): a blank group, a control group, and a test group, each with six replicates. The total volume of each sample was 40 ml. The entire experiment was conducted in a waterbath at 39°C, simulating the rumen environment. The fermentation system was established in a culture bottle containing 0.5 g of fermentation substrate. Immediately after establishment, CO2 was introduced for 3–5 seconds to displace the air. The bottle was then sealed with a rubber stopper, and a 60 ml syringe was connected to the stopper via a two-way valve. All connections were sealed with tape to prevent air leaks. The culture bottle was then placed in a constant-temperature waterbath for in vitro fermentation. After fermentation, the fermentation broth was filtered and stored at −80°C until further use.

[0087] Table 6 Test group system

[0088]

[0089] Note: The DNA in the PEG group is Cas9-pC2A-hdrE.

[0090] 6. Methane gas analysis

[0091] Gas chromatography was used to determine the composition of the gases collected by syringe and analyze the percentage of methane during in vitro fermentation. The gas yield of each component was calculated using the following formula: Gas yield = Total gas production over 24 hours × Percentage of that component. The total gas production over 24 hours was calculated by reading the scale. Nitrogen was used as the carrier gas, with a flow rate of 20 ml / min, an injection volume of 1 ml, a column oven temperature of 80°C, and a thermal cell temperature of 150°C. Calculations were performed using the external standard method.

[0092] 7. Determination of pH value of fermentation broth

[0093] The pH value of the filtrate was measured after calibrating the pH meter (Sartorius PB-10) according to GB / T41439-2022. Under repeatability conditions, the absolute difference between two independent measurements should not exceed 0.2.

[0094] 8. Data Processing

[0095] GraphPad Prism 10.1.2 was used for graph drawing, and SPSS 26.0 software was used for analysis of the experimental data. P < 0.05 was considered a significant difference, P < 0.01 was considered a very significant difference, and P < 0.001 was considered an extremely significant difference.

[0096] 9. Results

[0097] See the results Figure 4-6 And Table 7. Figure 4is the total gas production during in vitro fermentation, Figure 5 is the ratio of methane gas produced in the in vitro fermentation process to the total gas production, Figure 6 is the pH value of the fermentation liquid after 24 hours of in vitro fermentation; in the figure, * indicates that there is a significant difference between the two groups, ** indicates that there is a very significant difference between the two groups, and *** indicates that there is an extremely significant difference between the two groups.

[0098] Table 7 Effects of different treatments on methane production and pH value

[0099]

[0100] from Figure 4-6 As can be seen from Table 7, after the targeted editing system of the hdrE gene of Methanosarcina was introduced into the transformation 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 shows that the method used in the present invention has a significant effect in reducing methane emissions.

[0101] Furthermore, the results showed that the pH of the rumen fluid exhibited a certain degree of change after treatment. Notably, this change remained within the pH range of normal rumen fermentation in cattle. Combined with the results of rumen microbial qPCR analysis, it can be seen that the method adopted by the present invention effectively reduced the relative abundance of methanogens, thereby adjusting the microbial community structure in the in vitro fermentation system. From a macroscopic perspective, this adjustment affected the acid-base balance of the fermentation environment but did not disrupt the normal physiological function of the rumen, thus ensuring the overall stability of the rumen fermentation process.

[0102] Example 3 Analysis of Rumen Microorganisms in In Vitro Fermentation

[0103] DNA was extracted from the stored fermentation broth using the EZNA® Soil DNA Kit according to the manufacturer's instructions. The concentration and purity of the extracted genomic DNA were assessed by measuring the A260 / 280 ratio and verified on a 1% agarose gel. DNA samples were diluted to 10 ng / μL, and the relative abundance of rumen microorganisms was quantified using qPCR. The microorganisms analyzed included Bifidobacterium, Methanogens, Ruminococcus albus, Lactobacillus, Bacillus, and Actinobacillus succinogenes, and their relative abundance was calculated.

[0104] qPCR amplification reactions were performed in a 20 μL system containing 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 procedure 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 for the target bacteria, Ct total bacteria is the Ct value obtained using the total bacterial primers.

[0105] See Table 8 for specific primer sequences used for qPCR.

[0106] Table 8 Specific primer sequences for qPCR

[0107]

[0108] The effects of different treatment groups on microorganisms in the in vitro fermentation system are shown in Table 9 and Figure 7 As shown, Figure 7 The content of each microorganism was compared with the total bacteria as a reference. The specific microbial species measured were Bifidobacterium ×10 -3 ), Methanogens ×10 -3 )、Ruminococcus albus ×10 -4 )、Lactobacillus ×10 -1 ), Bacillus ×10⁻³, and Actinobacillus succinogenes ×10⁻².

[0109] Table 9 Effects of different treatment groups on microorganisms in the in vitro fermentation system

[0110]

[0111] Compared to the control and blank groups, the abundance of specific microorganisms in the experimental groups during in vitro rumen fermentation significantly changed. Specifically, when the targeted editing system for the hdrE gene of Methanosarcina was introduced into the transformation mixture, the abundance of methanogens was significantly reduced (P ≤ 0.001). This change demonstrates that the method can effectively inhibit the growth of methanogens, thereby reducing methane production and achieving the goal of reducing methane emissions.

[0112] Significant changes in the abundance of other microorganisms were also observed, with Ruminococcus albus, Bacillus, and Bacillus all showing fluctuations in abundance. This phenomenon may be due to the different conversion systems affecting the microbial communities in the original rumen fluid, which manifests itself at the macro level as changes in the pH of the fermentation broth.

Claims

1. An in vitro evaluation method for the effect of a targeted editing system of the hdrE gene of Methanosarcina 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) The targeted editing system of the Methanosarcina hdrE gene described in step (1) is coated into PLL-PEG-DNA nanoparticles using a PLL-PEG polymer, and the nanoparticles are mixed with bovine rumen fluid, silage, and a conversion 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: relative abundance of Methanosarcina, methane gas production, and changes in rumen microbial community structure; 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. The in vitro evaluation method according to claim 1, wherein 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.

3. The in vitro evaluation method according to claim 1, wherein The transformation mixture contains: 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.

4. The in vitro evaluation method according to claim 2, wherein Sodium hydroxide was added dropwise to adjust the pH of the conversion mixture to 7.

4.

5. The in vitro evaluation method according to claim 1, wherein In the step (3), the culture time is 24 hours, and the effect is evaluated using the proportion of methane in the generated gas as an evaluation index.

6. Use of the in vitro evaluation method according to claim 1, characterized in that: Used to regulate cattle rumen methane emissions.

7. Use of the in vitro evaluation method according to claim 1, characterized in that: Used to prepare products that reduce methane production in the rumen of cattle.

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