Gene expression inhibition system independent of plant endogenous factors and application of gene expression inhibition system in plants

Abstract

The invention discloses a tool for realizing efficient specific gene inhibition in a plant by utilizing a SunTag system of CRISPR-dCas9 (clustered regularly interspaced short palindromic repeats-das9). According to the method, gRNA near a transcription start site of a targeted target gene is designed, dCas9 and scFv-GFP are accurately recruited to a target site by using a SunTag system, the transcription start process of RNA polymerase II is interfered, and the expression of the target gene is remarkably inhibited. In view of relatively high activity of the system in a plant rdr6 mutant, an artificial miR-RDR6 fragment is further introduced into a vector and is used for inhibiting expression of RDR6 in a wild plant, so that the applicability and functionality of the system are expanded. Compared with a traditional method depending on a plant endogenous effector, the method has the advantages that dependence on endogenous regulatory factors is avoided, the specificity and safety of gene inhibition are remarkably improved, and the method can be used for plant genetic function research and character improvement.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to a tool and method for achieving efficient and specific inhibition of target gene expression in plants using the CRISPR-dCas9-SunTag system, and in particular to a gene expression inhibition system that does not rely on endogenous plant regulatory factors and its application in plant genetic function research and trait improvement. Background Technology

[0002] Precise gene regulation often plays a decisive role in important agronomic traits of crops, such as disease resistance, stress resistance, yield, and quality. Precise gene regulation has a wide range of applications in molecular breeding of crops. In terms of gene expression suppression, the traditional tool for suppressing plant genes is RNA interference, which produces a gene silencing effect by expressing double-stranded RNA or artificial miRNA targeting the gene. However, the efficiency and specificity of this tool need improvement. In recent years, the discovery of the CRISPR-Cas system has not only revolutionized gene editing technology but also greatly promoted the development of gene regulation technology. The nuclease-inactivated CRISPR-dCas9 system can serve as a gene targeting tool, precisely binding to target sites under the guidance of gRNA. By fusing dCas9 with transcription factors or epigenetic effector proteins, this system can activate or inhibit the expression of target genes at the target site; these techniques are respectively called CRISPRa (CRISPR activation tool) and CRISPRi (CRISPR interference tool). Unlike traditional CRISPR-Cas9-mediated gene editing, CRISPRa or CRISPRi do not cause permanent changes to the DNA sequence, thus offering greater safety and reversibility.

[0003] However, the existing CRISPRa / i still faces some challenges. First, this tool relies on the high expression of exogenous effector proteins. Since these effector proteins have broad gene regulatory functions, their overexpression is likely to produce off-target effects, reducing their specificity. Post-transcriptional gene silencing (PTGS) is a defense mechanism discovered in the last decade for biological (especially eukaryotic) cells to resist the invasion of foreign nucleic acids and maintain the integrity of their own genome. It refers to a regulatory mechanism in which plants block gene expression after the transcription of foreign genes by degrading mRNA or inhibiting its translation. Second, in plants such as Arabidopsis thaliana, the expression efficiency of core elements such as dCas9 is low due to the limitations of the post-transcriptional gene silencing mechanism, thus limiting its regulatory effect. Summary of the Invention

[0004] To address the limitations of existing CRISPRi technologies, which rely on exogenous effector proteins and are constrained by RDR6-mediated post-transcriptional gene silencing (PTGS) mechanisms in plants, this invention provides a CRISPRi gene expression inhibition system that does not depend on exogenous effector proteins and its application in plants. Based on eukaryotic transcriptional regulation mechanisms, this system uses precisely designed guide RNAs to direct the dCas9 and scFv-GFP fusion protein complex to the transcription initiation region of the target gene, interfering with the normal binding and initiation of RNA polymerase II in this region, thereby achieving efficient and specific inhibition of target gene expression. Furthermore, given the crucial role of the RDR6 gene in the plant PTGS pathway, the inventors found that this CRISPRi system only exhibits good inhibitory effects in the rdr6 mutant background. To overcome this limitation, this invention integrates a synthetically produced miRNA element (amiR-RDR6) targeting the RDR6 gene into the vector, enabling it to effectively reduce RDR6 expression in a wild-type plant background, thereby relieving RDR6-mediated inhibition of dCas9 expression and ensuring the normal function of the CRISPRi system.

[0005] This invention provides the following technical solution:

[0006] First, the present invention provides a CRISPR-dCas9-SunTag-amiR-RDR6 vector that does not depend on endogenous plant regulatory factors. The vector includes a dCas9 expression cassette, an antibody fusion protein expression cassette, and a guide RNA expression cassette that expresses a target gene. The vector further includes a synthetically produced microRNA element (amiR-RDR6) that targets the RDR6 gene.

[0007] In a preferred embodiment, the dCas9 expression cassette comprises, from the 5′ end to the 3′ end, a promoter for driving dCas9 expression, a nuclease-inactivated Cas9 (dCas9) coding sequence, and a GCN4 antigen peptide sequence with 10 tandem repeats; the antibody fusion protein expression cassette comprises, from the 5′ end to the 3′ end, a promoter, an scFv antibody fragment coding sequence, and a green fluorescent protein (GFP) coding sequence.

[0008] In a more preferred embodiment, the artificially synthesized microRNA sequence targeting the RDR6 gene is shown in SEQ ID NO.4.

[0009] In addition, the present invention provides a method for inhibiting the expression of a target gene in plants, the method comprising introducing the CRISPR-dCas9-SunTag-amiR-RDR6 vector of claim 1 into plant cells.

[0010] In one embodiment, the plant is a wild-type plant, and the microRNA element targeting the RDR6 gene in the CRISPR-dCas9-SunTag-amiR-RDR6 vector is used to reduce the expression of the endogenous RDR6 gene in the plant.

[0011] In a preferred embodiment, the method includes the following steps: S1, transferring the vector into Agrobacterium tumefaciens strain and culturing and screening to obtain positive bacterial solutions; S2, immersing the plant in full bloom in the Agrobacterium tumefaciens bacterial solution for 1 minute, culturing in the dark for 10 hours, then transferring to conventional light conditions for continued culturing for about 3 to 4 weeks, and harvesting T1 generation seeds;

[0012] S3. After surface disinfection, the T1 generation seeds are sown in a culture medium containing a screening agent for screening. After an appropriate culture time, the expression level of the target gene is detected to evaluate the gene suppression effect.

[0013] In a more preferred embodiment, the plant is Arabidopsis thaliana.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] 1. The CRISPRi system of the present invention can suppress the expression of endogenous genes in plants without relying on additional exogenous transcriptional effector proteins, thus avoiding non-specific effects caused by overexpression of effector proteins and improving the specificity and safety of suppression;

[0016] 2. By integrating the amiR-RDR6 element into the CRISPRi system, the restriction of dCas9 expression by the RDR6-mediated PTGS mechanism was effectively overcome. The constructed CRISPRi-amiR-RDR6 tool also showed efficient and stable gene repression in wild-type plants, significantly expanding its application scope in the field of plant gene regulation and genetic improvement.

[0017] 3. This system can precisely inhibit the expression of target genes while causing less interference with non-target genes, thereby reducing the risk of potential off-target effects. Attached Figure Description

[0018] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0019] Figure 1This invention provides a comparison of FWA gene expression levels and leaf number in the SunTag-CRISPRi system for suppressing the endogenous Arabidopsis gene FWA in Arabidopsis fwa rdr6 mutant, using T1 generation transgenic Arabidopsis and fwardr6 mutant.

[0020] Figure 2 This is a comparison of the expression levels of ABA2 and ELF8 genes and plant phenotypes in T1 generation transgenic Arabidopsis gABA2-SunTag, gELF8-SunTag and rdr6 in the SunTag-CRISPRi system used in this invention to suppress endogenous genes ABA2 and ELF8 in Arabidopsis thaliana, under the background of Arabidopsis thaliana rdr6 mutant.

[0021] Figure 3 This is a comparison of ABA2 gene expression in T1 generation transgenic Arabidopsis and wild-type Arabidopsis in the CRISPRi-amiR-RDR6 system used in this invention to suppress the endogenous Arabidopsis gene ABA2, under a wild-type Arabidopsis background.

[0022] Figure 4 This invention relates to the SunTag-CRISPRi system for inhibiting the endogenous Arabidopsis gene FWA, specifically the binding of Pol2 at the promoter site of the FWA gene in T1 generation transgenic Arabidopsis and the Fwardr6 mutant in the Arabidopsis fwa rdr6 mutant background. Detailed Implementation

[0023] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0024] The SunTag-CRISPRi system used in this invention is disclosed in U.S. Patent Application Publication US20240141367A1, the relevant disclosure of which is hereby incorporated by reference. Specifically, the system includes a nuclease-inactivating Cas9 protein (dCas9) carrying 10 GCN4 polypeptide tags and a synergistic scFv-GFP fusion protein, used to recruit multiple copies of the protein complex in the promoter region of the target gene, thereby interfering with the transcription initiation of RNA polymerase and achieving the purpose of efficiently inhibiting gene expression.

[0025] Example 1: Suppression of the FWA gene in the fwa-rdr6 double mutant Arabidopsis thaliana

[0026] RDR6 (RNA-Dependent RNA Polymerase 6) is a plant-specific RNA-dependent RNA polymerase. It transcribes single-stranded RNA (ssRNA) into double-stranded RNA (dsRNA), a crucial step in initiating the PTGS reaction. Overexpressed exogenous genes in plants are often silenced by the RDR6-mediated PTGS mechanism, affecting transgene expression. Therefore, using rdr6 mutant plants can significantly improve editing efficiency.

[0027] To achieve the effect of reducing endogenous gene expression in plants without relying on exogenous effector proteins, this invention first conducted experiments in the Arabidopsis fwa-rdr6 double mutant, in which the FWA gene is highly expressed, while the key PTGS gene RDR6 is suppressed. The experimental steps are as follows:

[0028] S1. The target gene in this embodiment is FWA, and the sequence of the guide RNA is shown in SEQ ID NO.1. After constructing the FWA gRNA into the SunTag-CRISPRi vector, it is transformed into Agrobacterium. The transformation method is as follows: 200 ng of plasmid is taken into an EP tube, Agrobacterium competent cells are added, mixed, and then placed on ice for 20 minutes. Then, it is incubated in liquid nitrogen for 1 minute, placed at 37°C for 2 minutes, placed on ice for 2-3 minutes, and then added to antibiotic-free LB medium. After recovery on a shaker at 28°C and 250 rpm / min for 2 hours, it is plated onto a Cannab-Rifampicin resistance plate for selection for 2-3 days.

[0029] S2. The method of infecting Arabidopsis thaliana by dipping flowers is as follows: After the positive Agrobacterium monoclonal expansion culture is carried out, the Agrobacterium infection solution is obtained by resuspending it in 5% sucrose and 0.02% Silwet77. Arabidopsis thaliana in full bloom is immersed in the infection solution for 1 minute, cultured in the dark for 10 hours, and then cultured under light for about 3-4 weeks to harvest T1 generation seeds.

[0030] S3. Plant selection method: After disinfecting T1 generation seeds, they were screened on 1 / 2 MS plates containing hygromycin. After 10 days of growth, the healthy positive seedlings were selected and transplanted into the soil. After 2 weeks of culture, the expression of FWA gene was detected and the number of flowering leaves of Arabidopsis thaliana was counted.

[0031] The results are as follows Figure 1 As shown in the quantitative fluorescence results, the FWA gene was successfully suppressed by approximately 90% in the transgenic plants obtained in this example. Phenotypic observation also showed that the flowering time of the transgenic plants was earlier than that of the control group (a clear late-flowering phenotype was observed). This indicates that the FWA gene can be successfully suppressed in the rdr6 mutant through CRISPRi.

[0032] Example 2: Repression of ABA2 and ELF8 genes in rdr6 single mutant Arabidopsis thaliana

[0033] In this embodiment, we aim to further explore whether the system can function on other endogenous genes. The plant selected in this embodiment is the rdr6 single mutant Arabidopsis thaliana, and the experimental steps are as follows:

[0034] S1. ABA2 and ELF8 were selected as target genes, and their guide RNA sequences are shown in SEQ ID NO.2 and 3, respectively. The gRNA was introduced into the SunTag-CRISPRi vector and then transformed into Agrobacterium. The transformation method was as follows: 200 ng of plasmid was aspirated into an EP tube, Agrobacterium competent cells were added, the mixture was mixed, and the cells were incubated on ice for 20 minutes. Then, the cells were incubated in liquid nitrogen for 1 minute, placed at 37°C for 2 minutes, and incubated on ice for 2-3 minutes. Antibiotic-free LB medium was added, and the cells were incubated on a shaker at 28°C and 250 rpm / min for 2 hours. The cells were then plated onto Cannab-Rifampicin resistance plates for selection for 2-3 days.

[0035] S2. The method of infecting Arabidopsis thaliana by dipping flowers is as follows: After the positive Agrobacterium monoclonal expansion culture is carried out, the Agrobacterium infection solution is obtained by resuspending it in 5% sucrose and 0.02% Silwet77. Arabidopsis thaliana in full bloom is immersed in the infection solution for 1 minute, cultured in the dark for 10 hours, and then cultured under light for about 3-4 weeks to harvest T1 generation seeds.

[0036] S3. Plant selection: The method is as follows: After disinfecting the T1 generation seeds, they were screened on 1 / 2 MS plates containing hygromycin. After growing for 10 days, the healthy positive seedlings were selected and transplanted into the soil. After culturing for 2 weeks, the expression of ELF8 and ABA2 genes was detected and the number of flowering leaves of Arabidopsis thaliana was counted.

[0037] The results of this embodiment are as follows: Figure 2 As shown, in an rdr6 background, quantitative fluorescence results indicated that the ABA2 and ELF8 genes were successfully suppressed by approximately 40-50% in the transgenic plants obtained in this example, and the corresponding plants also exhibited mutant phenotypes of dwarfism and early flowering, respectively. This demonstrates that the rdr6 single mutant system can effectively relieve the suppression of dCas9 expression in the CRISPRi system, ensuring the normal function of the CRISPRi system.

[0038] Example 3: Repression of ABA2 and ELF8 genes in wild-type Arabidopsis thaliana

[0039] Based on the results of Examples 1 and 2, we constructed a SunTag-CRISPRi-amiR-RDR6 system and verified its gene-targeting repression function in wild-type plants. The experimental steps are as follows:

[0040] S1. Carrier Construction:

[0041] First, the original SunTag-CRISPRi vector was linearized using the restriction endonuclease Pme1. The DNA sequence of the artificial miRNA target RDR6 gene is shown in SEQ ID NO:4. Then, amiR-RDR6 was tandemly ligated into the linearized SunTag-CRISPRi vector using homologous recombination to construct SunTag-CRISPRi-amiR-RDR6.

[0042] S2. The target gene in this embodiment is ABA2. The sequence of the guide RNA is shown in Table 1. The gRNA was introduced into the SunTag-CRISPRi-amiR-RDR6 vector and then transformed into Agrobacterium. The transformation method is as follows: 200 ng of plasmid was taken into an EP tube, Agrobacterium competent cells were added, mixed and placed on ice for 20 minutes, then incubated in liquid nitrogen for 1 minute, placed at 37°C for 2 minutes, placed on ice for 2-3 minutes, antibiotic-free LB medium was added, and the cells were thawed on a shaker at 28°C and 250 rpm / min for 2 hours. The cells were then plated onto Cannab-Rifampicin resistance plates for selection for 2-3 days.

[0043] Table 1 Guide RNA Sequence

[0044]

[0045] S3. The method of infecting Arabidopsis thaliana by dipping flowers is as follows: After the positive Agrobacterium monoclonal expansion culture is carried out, the Agrobacterium infection solution is obtained by resuspending the culture in 5% sucrose and 0.02% Silwet77. Arabidopsis thaliana in full bloom is immersed in the infection solution for 1 minute and cultured in the dark for 10 hours. After that, the seeds of the T1 generation are harvested after about 3-4 weeks of culture under light.

[0046] S4. Plant selection: The method is as follows: After disinfecting the T1 generation seeds, they are screened on 1 / 2 MS plates containing hygromycin. After growing for 10 days, the healthy positive seedlings are selected and transplanted into the soil. After culturing for 2 weeks, the expression of the ABA2 gene is detected and the phenotype is observed.

[0047] The results are as follows Figure 3 As shown, ABA2 gene expression was successfully suppressed in the wild-type background. Further to illustrate the suppression mechanism of SunTag-CRISPRi, ChIP-seq of Pol II was used to analyze the binding of Pol II at the promoter site of the T1 generation SunTag-CRISPRi transgenic Arabidopsis and the FWA gene of the Fwardr6 mutant in the Fwardr6 background of Example 1. The results are as follows: Figure 4 The results show that the SunTag-CRISPRi system can interfere with the binding of PolII in the gene promoter region, thereby inhibiting gene expression.

[0048] Therefore, it can be seen that the SunTag-CRISPRi-amiR-RDR6 system we constructed based on SunTag-CRISPRi can stably suppress endogenous genes in plants.

[0049] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A CRISPR-dCas9-SunTag-amiR-RDR6 vector independent of plant endogenous regulatory factors, characterized in that, The vector includes a dCas9 expression cassette, an antibody fusion protein expression cassette, and a guide RNA expression cassette that expresses a target gene; the vector further includes a synthetically produced microRNA element (amiR-RDR6) that targets the RDR6 gene.

2. The carrier according to claim 1, characterized in that, The dCas9 expression cassette, from 5′ to 3′, comprises: a promoter for driving dCas9 expression, a nuclease-inactivated Cas9 (dCas9) coding sequence, and a GCN4 antigen peptide sequence with 10 tandem repeats; the antibody fusion protein expression cassette, from 5′ to 3′, comprises: a promoter, an scFv antibody fragment coding sequence, and a green fluorescent protein (GFP) coding sequence.

3. The carrier according to claim 1 or 2, characterized in that, The artificially synthesized microRNA sequence targeting the RDR6 gene is shown in SEQ ID NO.

4.

4. A method for inhibiting the expression of a target gene in plants, characterized in that, The method includes introducing the CRISPR-dCas9-SunTag-amiR-RDR6 vector of claim 1 into plant cells.

5. The method according to claim 4, characterized in that, The plant is a wild-type plant, and the microRNA element targeting the RDR6 gene in the CRISPR-dCas9-SunTag-amiR-RDR6 vector is used to reduce the expression of the endogenous RDR6 gene in the plant.

6. The method according to claim 4 or 5, characterized in that, The method includes the following steps: S1. The vector was transferred into Agrobacterium tumefaciens strain and cultured and screened to obtain positive bacterial solutions; S2. Immerse the plant in full bloom in the Agrobacterium tumefaciens solution for 1 minute, culture in the dark for 10 hours, then transfer to normal light conditions and continue culture for about 3 to 4 weeks to harvest T1 generation seeds; S3. After surface disinfection, the T1 generation seeds are sown in a culture medium containing a screening agent for screening. After an appropriate culture time, the expression level of the target gene is detected to evaluate the gene suppression effect.

7. The method according to claim 6, characterized in that, The plant in question is Arabidopsis thaliana.

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