Method for regulating methylation level of specific region of plant genomic DNA using Cas12i system

Abstract

The present application relates to the technical field of biotechnology, and particularly relates to a method for regulating methylation level of specific region of plant genome DNA using Cas12i system. A fusion protein is provided, which comprises, from N-terminal to C-terminal, a first nuclear localization signal peptide, a methylation modification domain, a connecting peptide, a Cas12i protein without nuclease activity and a second nuclear localization signal peptide; the methylation modification domain is an OsSUVH2 protein capable of up-regulating methylation level or a functionally active fragment thereof; the amino acid sequence of the OsSUVH2 protein is shown as SEQ ID NO:1; and the amino acid sequence of the Cas12i protein without nuclease activity is shown as SEQ ID NO:3. A CRISPR-dCas12i system constructed based on the fusion protein is also provided. The CRISPR-dCas12i system significantly improves the methylation modification efficiency of plant genome DNA.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a method for regulating the methylation level of specific regions of plant genomic DNA using the Cas12i system. Background Technology

[0002] DNA methylation is a conserved and crucial epigenetic modification mechanism in which organisms regulate gene expression by chemically modifying bases to methylate without altering the DNA sequence itself. In higher plants, DNA methylation primarily occurs at the fifth carbon atom of cytosine (5mC), and three main sequence backgrounds exist: CG, CHG, and CHH (where H represents A, T, or C). This modification plays a vital role in maintaining genome stability, regulating spatiotemporally specific gene expression, silencing transposon elements, imprinted gene expression, and responding to biotic and abiotic stresses. Notably, some DNA methylation states are transgenerationally inheritable, forming stable epialeleles and thus producing heritable phenotypic variations. Classic examples include the methylation status of the FLOWERING WAGENINGEN (FWA) gene in Arabidopsis thaliana, which affects flowering time (Gallego-Bartolomé, Gardiner et al. 2018), and the Epi-d1 site in rice, which controls plant architecture and yield (Miura, Agetsuma et al. 2009). These findings demonstrate that precise regulation of DNA methylation is not only crucial for understanding fundamental plant biological processes but also provides a new dimension for crop genetic improvement.

[0003] Rice (Oryza sativa), as an important food crop and a model monocotyledonous plant, has a large genome with a complex methylation pattern. The uniqueness and complexity of its DNA methylation pattern are mainly reflected in the following aspects: Higher overall methylation level: The overall methylation levels of CG, CHG, and CHH in the rice genome are significantly higher than in Arabidopsis, suggesting that methylation plays a more extensive and profound role in the regulation of the rice genome. Complex spatial distribution pattern: In rice, CG and CHG methylation are mainly enriched in heterochromatin regions near the centromere and on long transposable elements (TEs), primarily responsible for maintaining chromatin structure and silencing TEs. In contrast, CHH methylation is more widely distributed in euchromatin regions, especially on numerous miniature inverted repeat transposable elements (MITEs) in intergenic regions and promoter regions. The dynamic changes in CHH methylation on these MITEs close to genes are considered key switches for finely regulating the expression of neighboring genes. Extensive Gene Region Methylation Modification: Studies have shown that methylation modifications exist in the promoters or coding regions of numerous functional genes in rice, including many key genes involved in seed development, organogenesis, flowering regulation, and stress resistance. For example, the imprinted expression of the OsFIE1 gene is strictly regulated by DNA methylation. Although we recognize the importance of DNA methylation in rice, our understanding of how specific methylation states at most gene loci affect gene expression, the synergistic mechanisms between different types of methylation, and how to precisely manipulate these modifications remains very limited. This significantly restricts our ability to utilize epigenetic variation in rice molecular breeding.

[0004] To elucidate the function of DNA methylation at specific sites, researchers urgently need tools capable of site-specific erasure or creation of DNA methylation modifications. Traditional gene knockout or overexpression methods can only alter gene sequences or overall expression levels, failing to change their epigenetic state. In recent years, epigenetic editing technologies based on the CRISPR / Cas system have emerged, bringing revolutionary breakthroughs to this field. The basic principle of this technology is to utilize Cas proteins with inactivated nuclease activity (such as dCas9) as DNA-binding modules, fusing them with effector proteins that have DNA methylation modification (such as DNMT3A, SUVH9) or demethylation (such as TET1) functions. Guided by sgRNA, the effector proteins are specifically recruited to target genomic regions, thereby achieving site-specific epigenetic modifications. For example, early pioneering research fused the human demethylase TET1cd with dCas9, successfully achieving site-specific demethylation in the promoter region of the Arabidopsis FWA gene, activating gene expression and leading to a heritable late-flowering phenotype. Conversely, fusing key factors in the Arabidopsis RdDM (RNA-mediated DNA methylation) pathway with artificial zinc finger proteins or dCas9 can increase the methylation level at target sites and inhibit gene expression. We have also successfully constructed a fusion protein of dCas9 and rice OsSUVH2 and successfully upregulated the DNA methylation level of the promoter region of the rice ideal plant architecture gene IPA1 (Patent No.: ZL202110046860.9).

[0005] However, despite the success of dCas9-based tools in Arabidopsis and some animal cells, applying them to complex crop genomes such as rice still faces numerous challenges: First, the classic SpCas9 requires a strict "NGG" pre-intergenic region sequence neighbor motif (PAM), which greatly limits its targetable range in AT-rich plant promoters or intergenic regions (such as MITEs). Second, the Cas9 protein itself has a large molecular weight (approximately 160 kDa), and when fused with large epigenetic modifying enzymes (such as TET1 complexes), the entire complex becomes too bulky, making it difficult to efficiently enter the plant cell nucleus, and may be affected by chromatin steric hindrance, leading to low editing efficiency. Finally, non-specific binding can still lead to changes in the epigenetic state of non-target regions, causing unpredictable side effects.

[0006] Given the importance and complexity of DNA methylation regulation in rice, and the limitations of existing dCas9 tools in rice applications, there is an urgent need to develop a more efficient, precise, and broadly targeted methylation editing tool based on novel chassis proteins. Summary of the Invention

[0007] To overcome the limitations of traditional dCas9 editing systems in targeting AT-rich regions (such as promoters and MITEs regions) in rice (due to NGG PAM) and the difficulty of large protein complexes entering the nucleus, this invention provides a fusion protein for site-specific regulation of methylation levels in specific regions of plant genomic DNA (especially AT-rich regions), the CRISPR-dCas12i system, and its application in crop genetic improvement.

[0008] In a first aspect, the present invention provides a fusion protein comprising, from the N-terminus to the C-terminus, a first nuclear localization signal peptide, a methylation-modified domain, a linker peptide, a nuclease-free Cas12i protein, and a second nuclear localization signal peptide; the methylation-modified domain is an OsSUVH2 protein or a functionally active fragment thereof capable of upregulating methylation levels; the amino acid sequence of the OsSUVH2 protein is shown in SEQ ID NO: 1; the amino acid sequence of the nuclease-free Cas12i protein is shown in SEQ ID NO: 3.

[0009] The nuclease-free Cas12i protein (also referred to herein as dCas12i3-5M or dCas12i3) loses its ability to cleave double-stranded DNA while retaining its DNA-binding activity.

[0010] In some embodiments, the amino acid sequence of the linker peptide is shown in SEQ ID NO: 15 or SEQ ID NO: 16.

[0011] In some embodiments, the amino acid sequence of the first nuclear localization signal peptide is shown in SEQ ID NO: 18, and the amino acid sequence of the second nuclear localization signal peptide is shown in SEQ ID NO: 18 or SEQ ID NO: 19.

[0012] In some embodiments, the amino acid sequence of the fusion protein is shown in SEQ ID NO: 22.

[0013] The fusion protein, with the amino acid sequence shown in SEQ ID NO: 22, has the following structure from the N-terminus to the C-terminus: SV40NLS-OsSUVH2-1×GGGGS-XTEN-dCas12i3-5M-NucleoplasminNLS. The OsSUVH2 protein and the nuclease-free Cas12i protein (dCas12i3-5M) are linked by a linker peptide (1×GGGGS-XTEN) with the amino acid sequence shown in SEQ ID NO: 15. The N-terminus contains the nuclear localization signal peptide SV40NLS with the amino acid sequence shown in SEQ ID NO: 18, and the C-terminus contains the nuclear localization signal peptide NucleoplasminNLS with the amino acid sequence shown in SEQ ID NO: 19. The linker peptide (1×GGGGS-XTEN) balances the flexibility and rigidity of the domains at both ends of the fusion protein, significantly improving methylation modification efficiency.

[0014] In a second aspect, the present invention provides a product (i.e., a CRISPR-dCas12i system) for increasing the methylation level of specific regions of plant genomic DNA, said product comprising any one of the following (a)-(e):

[0015] (a) The fusion protein and the guide RNA;

[0016] (b) An expression construct 1 comprising the nucleotide sequence encoding the fusion protein, and a guide RNA;

[0017] (c) The fusion protein, and expression construct 2 containing a nucleotide sequence encoding a guide RNA;

[0018] (d) An expression construct 1 containing a nucleotide sequence encoding the fusion protein, and an expression construct 2 containing a nucleotide sequence encoding a guide RNA;

[0019] (e) An expression construct 3 comprising a nucleotide sequence encoding the fusion protein and a nucleotide sequence encoding the guide RNA;

[0020] The guide RNA comprises:

[0021] (i) spacer sequences capable of hybridizing with the target sequence in the specific region, and

[0022] (ii) A direct repeat sequence attached to the spacer sequence; the direct repeat sequence is capable of guiding the fusion protein to bind to the guide RNA to form a CRISPR-Cas complex targeting the target sequence.

[0023] The specific region refers to the target sequence and its surrounding area.

[0024] In some embodiments, in expression construct 1 and expression construct 3, the promoter for initiating transcription of the nucleotide sequence encoding the fusion protein is the Ubi promoter.

[0025] The Ubi promoter can be a maize ubiquitin promoter with a nucleotide sequence as shown in SEQ ID NO: 5.

[0026] In some embodiments, in expression construct 2 and expression construct 3, the promoter for initiating transcription of the nucleotide sequence encoding the guide RNA is the U3 promoter.

[0027] The U3 promoter can be the rice U3 promoter with a nucleotide sequence as shown in SEQ ID NO: 6.

[0028] In some embodiments, the nucleotide sequence encoding the direct repeat sequence is shown in SEQ ID NO: 7.

[0029] Thirdly, the present invention claims protection for the use of the product described in the second aspect in increasing the methylation level of specific regions of plant genomic DNA. The use includes the step of introducing the product into a recipient plant to obtain a transgenic plant.

[0030] The product can be introduced into the recipient plant using any suitable method, including: transforming plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated transformation, and culturing the transformed plant tissues into plants.

[0031] After the product is introduced into the recipient plant, the guide RNA targets the fusion protein to the target sequence in the genomic DNA of the recipient plant, and the methylation modification domain in the fusion protein increases the methylation level of the target sequence and the surrounding region.

[0032] The transgenic plants include first-generation transgenic plants and their offspring, as well as plants obtained by crossing first-generation transgenic plants or their offspring with other varieties of the same species.

[0033] Fourthly, the present invention seeks protection for the use of the product described in the second aspect in plant breeding.

[0034] The plant breeding includes: introducing the product into a recipient plant to obtain a first plant with altered methylation levels in a specific region of the recipient plant's genomic DNA, and hybridizing the first plant with a second plant whose methylation levels in the specific region are not altered, thereby introducing the altered methylation levels in the specific region into the second plant.

[0035] The first plant and the second plant are hybridizable plants, preferably the same species.

[0036] In all of the above aspects, the plant may be a monocotyledonous plant, preferably a grass, and more preferably rice.

[0037] Fifthly, the present invention provides a method for increasing the methylation level of a specific region of rice genomic DNA, comprising: introducing the product described in the second aspect into rice, wherein the specific region is the IPA1 gene promoter region, and the target sequence is shown in SEQ ID NO: 10.

[0038] This invention utilizes the nuclease-free Cas12i protein (dCas12i3-5M) fused with the endogenous methylation key factor OsSUVH2 to develop a novel DNA methylation targeting modification system (CRISPR-dCas12i system) suitable for monocotyledonous plants such as rice. This system leverages Cas12i's specific recognition of T-rich PAM sequences (5'-TTN-3') to effectively target AT-rich promoter or transposon regions in the plant genome, overcoming the limitation of the Cas9 system in editing AT-rich regions (such as the key regulatory region of the IPA1 gene promoter), thus expanding the toolbox of DNA methylation regulation in plants. Utilizing the RNase activity inherent in Cas12i, this system requires only one promoter-driven crRNA array to achieve simultaneous editing of multiple sites, simplifying vector construction. During system development, systematic screening and optimization of the fusion direction (N-terminus vs. C-terminus) of dCas12i3-5M and OsSUVH2, the linker peptide, and the nuclear localization signal (NLS) significantly improved the system's activity in regulating DNA methylation. Experiments demonstrated that the optimized CRISPR-dCas12i system exhibited significantly higher methylation modification efficiency in rice than the existing CRISPR-dCas9 system. Figure 8 ).

[0039] The fusion protein and CRISPR-dCas12i system provided by this invention solve the problems of nuclear entry and stability of macromolecular fusion proteins in plant cells in the existing CRISPR-dCas9 system, and significantly improve the efficiency of DNA methylation modification, providing strong technical support for in-depth analysis of crop epigenetic regulatory mechanisms and creation of new epigenetic germplasm resources with excellent agronomic traits. Attached Figure Description

[0040] Figure 1 : A schematic diagram of the structure of the CRISPR-dCas12i system building block.

[0041] Figure 2A schematic diagram showing the positions of target sequence 1 (i.e., target point 1) and target sequence 2 (i.e., target point 2) in the promoter region of the rice IPA1 gene, and a comparison of PAM sequences.

[0042] Figure 3 Statistical results of methylation regulatory activities of constructs 1-4 in rice protoplasts (comparison of methylation editing efficiency of N-terminal and C-terminal fusion strategies of fusion proteins). Wild type: Rice Zhonghua 11; SUVH-dCas9+ target 1 crRNA: OsSUVH2-dCas9 vector; SUVH-dCas12i3+ target 2 crRNA: construct 1; dCas12i3-SUVH+ target 2 crRNA: construct 2; SUVH-dCas12i3 without crRNA: construct 3; dCas12i3-SUVH without crRNA: construct 4; Compared with the wild type of untransformed constructs, ***P<0.001, no significant difference in ns (Student's t-test).

[0043] Figure 4 Statistical results of methylation regulatory activities of constructs 5-8 in rice protoplasts (comparison of the effects of different types of linker peptides on methylation editing efficiency). No linker sequence: Construct 1; 3×GGGGS: Construct 5; 5×GGGGS: Construct 6; XTEN: Construct 7; 3×EAAAK: Construct 8; Compared with the no linker sequence, **P<0.01, ***P<0.001 (Student's t-test).

[0044] Figure 5 Statistical results of methylation regulatory activity of constructs 9-11 in rice protoplasts (effect of GGGGS and XTEN combined linker peptides on methylation editing efficiency). XTEN: construct 7; 1×GGGGS+XTEN: construct 9; 2×GGGGS+XTEN: construct 10; 3×GGGGS+XTEN: construct 11; Compared with XTEN, **P<0.01, ***P<0.001, no significant difference in ns (Student's t-test).

[0045] Figure 6 Statistical results of methylation regulatory activities of constructs 12-15 in rice protoplasts (the effect of the combination and position of different nuclear localization signal peptides on methylation editing efficiency). No NLS: Construct 9; SV-N_SV-C: Construct 12; Nu-N_Nu-C: Construct 13; SV-N_Nu-C: Construct 14; Nu-N_SV-C: Construct 15; Compared with no NLS, **P<0.01, ***P<0.001, no significant difference in ns (Student's t-test).

[0046] Figure 7 Gel electrophoresis image of PCR identification of transgenic positive plants of the construct.

[0047] Figure 8 Statistical results of methylation levels near target sequences in transgenic rice using the CRISPR-dCas12i system of this invention and the existing CRISPR-dCas9 system. Wild type: Rice Zhonghua 11; OsSUVH2-dCas9: OsSUVH2-dCas9 vector transgenic rice T0 generation plants; OsSUVH2-dCas12i3 without crRNA: Construct 16 transgenic rice T0 generation plants; OsSUVH2-dCas12i3+crRNA: Construct 14 transgenic rice T0 generation plants; Compared with OsSUVH2-dCas9, ***P<0.001 (Student's t-test).

[0048] Figure 9 Results of IPA1 gene expression level detection in transgenic rice. Wild type: Rice Zhonghua 11; OsSUVH2-dCas9: OsSUVH2-dCas9 vector transgenic rice T0 generation plants; OsSUVH2-dCas12i3 without crRNA: Construct 16 transgenic rice T0 generation plants; OsSUVH2-dCas12i3+crRNA: Construct 14 transgenic rice T0 generation plants; Compared with wild type, the IPA1 gene expression level of OsSUVH2-dCas9 was significantly reduced (Student's t-test, ***P<0.001); Compared with OsSUVH2-dCas9, the IPA1 gene expression level of OsSUVH2-dCas12i3+crRNA was significantly reduced (Student's st-test, ***P<0.001). Detailed Implementation

[0049] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Biochemical, molecular biological, genetic, and other related terms and laboratory procedures used herein are all widely used terms and routine procedures in their respective fields.

[0050] In this article, "Cas12i" or "Cas12i protein" refers to the class II type V CRISPR-Cas effector protein Cas12i and its variants (such as Cas12i1, Cas12i2, Cas12i3, etc.). Unlike Cas9, Cas12i typically recognizes T-rich PAM sequences (such as 5'-TTN-3') and possesses its own RNase activity to process crRNA arrays.

[0051] In this article, “dCas12i” or “dCas12i protein” refers to the catalytically inactivated Cas12i protein (dead Cas12i), whose RuvC domain contains a key amino acid mutation that causes it to lose its ability to cleave the DNA double helix, but retains its ability to specifically bind to target DNA.

[0052] In this paper, “OsSUVH2” refers to the rice endogenous lysine methyltransferase homolog SU(VAR)3-9HOMOLOG 2 (LOC_Os07g25450), which is a key recruitment factor in the DNA methylation pathway and can promote the establishment of DNA methylation modification.

[0053] The terms "guide RNA" and "gRNA" are used interchangeably in this article. In the CRISPR-dCas12i system described herein, the guide RNA is a man-made chimeric RNA molecule consisting of two parts: a spacer sequence responsible for recognizing and binding to the target DNA (target sequence), and a hairpin structure formed by direct repeat sequences (DR) responsible for recruiting and activating the Cas12 protein. The guide RNA precisely guides the Cas12i protein to a specific DNA target location.

[0054] "crRNA" in this article refers to CRISPR RNA, a guide RNA that contains a spacer sequence capable of hybridizing to a target sequence and a direct repeat sequence linked to said spacer sequence. The Cas12i system typically does not require tracrRNA; only crRNA is needed to guide the dCas12i complex to bind to the target site.

[0055] The rice variety “Zhonghua 11” is a japonica conventional rice variety, denoted as ZH11.

[0056] The standard recombinant DNA and molecular cloning techniques used in the following examples are well known to those skilled in the art and are described in more detail in the following literature: Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989.

[0057] The CRISPR single-base substitution vector pnCas9-PBE used in the following examples is described in the following literature: Zong, Yuan, Yanpeng Wang, Chao Li, Rui Zhang, Kunling Chen, Yidong Ran, Jin-LongQiu, Daowen Wang, and Caixia Gao. Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion. Nature biotechnology 35, no. 5(2017): 438.

[0058] The Agrobacterium EHA105 used in the following examples is disclosed in the literature Hood, EE; Gelvin, SB; Melchers, LS & Hoekema, A. (1993). "New Agrobacterium helper plasmids for gene transfer to plants". Transgenic Research. 2: 208-218. doi:10.1007 / BF01977351.

[0059] The present invention will now be described in detail with reference to specific embodiments.

[0060] Example 1: Regulating the methylation level of specific regions of rice genomic DNA using the CRISPR-dCas12i system

[0061] A CRISPR-dCas12i system was constructed to improve the methylation level of specific DNA sites and nearby regions in the plant genome, and the methylation modification efficiency of the system was verified by transient expression experiments in rice protoplasts and rice genetic transformation experiments.

[0062] I. Design of the Component

[0063] plasmid map of the construct, as shown Figure 1As shown in the diagram. In this construct, the rice endogenous lysine methyltransferase homolog SU(VAR)3-9 HOMOLOG 2 (LOC_Os07g25450) (i.e., OsSUVH2 protein) is fused with a Cas12i protein that has lost its DNA double-strand cleavage activity (i.e., dCas12i3-5M protein, also abbreviated as dCas12i3). The amino acid sequence of the OsSUVH2 protein is shown in SEQ ID NO: 1. The nucleotide sequence encoding the OsSUVH2 protein is shown in SEQ ID NO: 2. The amino acid sequence of the dCas12i3-5M protein is shown in SEQ ID NO: 3. The nucleotide sequence encoding the dCas12i3-5M protein is shown in SEQ ID NO: 4. The dCas12i3-5M protein is obtained by mutating the Cas12i3-5M protein (Cas-SF01, Duan et al., 2024, DOI: 10.1016 / j.xinn.2024.100564) with the E844A mutation (the glutamic acid at position 844 is mutated to alanine). Compared with the Cas12i3-5M protein, the dCas12i3-5M protein retains DNA-binding activity but loses DNA double-strand cleavage activity. The expression of the fusion protein is driven by the maize ubiquitin promoter (ZmUbi). The nucleotide sequence of the ZmUbi promoter is shown in SEQ ID NO: 5.

[0064] This construct also contains a Cas12i-specific crRNA expression cassette (abbreviated as Cas12i_crRNA), which includes a rice U3 promoter (OsU3 promoter), a nucleotide sequence encoding a Cas12i-specific direct repeat sequence (Cas12i3 DR), and a BsaI restriction site for inserting a nucleotide sequence encoding a spacer sequence in gRNA, enabling the construct to express crRNA targeting specific sites on genomic DNA. In the crRNA, the direct repeat sequence (Cas12i3 DR) guides the OsSUVH2-dCas12i3-5M fusion protein to bind to the crRNA to form a CRISPR-Cas complex, and the spacer sequence binds to the target sequence in genomic DNA, thereby regulating the methylation level of the target sequence and its surrounding region. The nucleotide sequence of the OsU3 promoter is shown in SEQ ID NO: 6. The nucleotide sequence encoding Cas12i3 DR is shown in SEQ ID NO: 7.

[0065] The original vector for this construct was a conventional CRISPR single-base substitution vector for rice transformation (pnCas9-PBE plasmid, described in the literature "Zong, Yuan, Yanpeng Wang, Chao Li, Rui Zhang, Kunling Chen, Yidong Ran, Jin-Long Qiu, Daowen Wang, and Caixia Gao. Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion. Nature biotechnology 35, no. 5 (2017): 438"). The construct was obtained by replacing the Cas9 protein-coding region in the original vector with the OsSUVH2-dCas12i3-5M fusion protein-coding region, and simultaneously replacing the original Cas9 sgRNA expression cassette with the Cas12i_crRNA expression cassette.

[0066] II. Preparation of the construct

[0067] 1. Introduce the dCas12i3-5M gene into the editing vector

[0068] The dCas12i3-5M gene, with its artificially synthesized nucleotide sequence as shown in SEQ ID NO: 4, was amplified by polymerase chain reaction (PCR) using primers dCas12i3-F / R. The nucleotide sequences of the primers are as follows:

[0069] dCas12i3-F: 5'-gtttggtgttacttctgcagcctaggatgaagaaggtcgaggtgag-3';

[0070] dCas12i3-R: 5'-cgatgatacgaacgaaagctctgagcttagagctccccggacgcgcttgactcctc-3'.

[0071] PCR was performed using KOD One™ PCR Master Mix (TOYOBO, KMM-101). The PCR system consisted of: 25 µL of KOD One™ PCR Master Mix (2x), 1.5 µL of dCas12i3-F (10 µM), 1.5 µL of dCas12i3-R (10 µM), 1 µL of the synthesized dCas12i3-5M gene, and PCR-grade pure water to a final volume of 50 µL. The PCR program was as follows: 98°C pre-denaturation for 1-2 min; 30 cycles of (98°C denaturation for 10 sec, 57°C annealing for 5 sec, 68°C extension for 10 sec); and a final extension at 68°C for 2 min. After the reaction, the PCR products were detected by agarose gel electrophoresis, and the dCas12i3-5M gene fragment was recovered by gel excision.

[0072] Using the pnCas9-PBE plasmid as a template, PCR was performed using primers ProUbi-F / R to amplify the maize ZmUbi promoter, whose nucleotide sequence is shown in SEQ ID NO: 5. The nucleotide sequences of the primers are as follows:

[0073] ProUbi-F: 5'-aaacgacggccagtgccaagcttggtacctgcagtgcagcgtgacccggt-3';

[0074] ProUbi-R: 5'-ctgcagaagtaacaccaaac-3'.

[0075] PCR system: KOD One™ PCR Master Mix (2x) 25 µL, ProUbi-F (10 µM) 1.5 µL, ProUbi-R (10 µM) 1.5 µL, pnCas9-PBE plasmid 1 µL, PCR-grade pure water to a final volume of 50 µL. PCR program: 98°C pre-denaturation for 1-2 min; (98°C denaturation for 10 sec, 57°C annealing for 5 sec, 68°C extension for 5 sec) 30 cycles; final extension at 68°C for 2 min. After the reaction, the PCR products were detected by agarose gel electrophoresis, and the corn ZmUbi promoter fragment was recovered by gel excision.

[0076] Using the pnCas9-PBE plasmid as a template, PCR was performed using primers TerE9-F / R to amplify the E9 terminator, whose nucleotide sequence is shown in SEQ ID NO: 24. The nucleotide sequences of the primers are as follows:

[0077] TerE9-F: 5'-cagagctttcgttcgtatcatc-3';

[0078] TerE9-R: 5'-aacagctatgacatgattacgaattcgttgtcaatcaattggcaag-3'.

[0079] PCR system: KOD One™ PCR Master Mix (2x) 25 µL, TerE9-F (10 µM) 1.5 µL, TerE9-R (10 µM) 1.5 µL, pnCas9-PBE plasmid 1 µL, PCR-grade pure water to a final volume of 50 µL. PCR program: 98°C pre-denaturation for 1-2 min; (98°C denaturation for 10 sec, 57°C annealing for 5 sec, 68°C extension for 5 sec) 30 cycles; final extension at 68°C for 2 min. After the reaction, the PCR products were detected by agarose gel electrophoresis, and the E9 terminator fragment was recovered by gel excision.

[0080] The recovered dCas12i3-5M gene fragment, ZmUbi promoter fragment, and E9 terminator fragment were used as templates to amplify the ZmUbi promoter-dCas12i3-5M gene-E9 terminator fusion fragment (referred to as the ZmUbi-dCas12i3-5M-E9 fragment) using primers dCas12i3-F and TerE9-R. PCR system: 25 µL KOD One™ PCR Master Mix (2x), 1.5 µL dCas12i3-F (10 µM), 1.5 µL TerE9-R (10 µM), 0.5 µL each of the three fragments, and PCR-grade pure water to a final volume of 50 µL. PCR program: 98°C pre-denaturation for 1-2 min; (98°C denaturation for 10 sec, 57°C annealing for 5 sec, 68°C extension for 15 sec) 30 cycles; 68°C final extension for 2 min. After the reaction, the PCR products were detected by agarose gel electrophoresis, and the ZmUbi-dCas12i3-5M-E9 fragment was recovered by gel excision.

[0081] The pnCas9-PBE plasmid was digested with restriction endonucleases HindIII (NEB, r3104) and EcoRI (NEB, r3101). The digestion products were detected by agarose gel electrophoresis, and the pnCas9-PBE linearized plasmid was recovered by gel excision. The recovered ZmUbi-dCas12i3-5M-E9 fragment was ligated into the pnCas9-PBE linearized plasmid using the ClonExpress II One Step Cloning Kit (Novizan, C112-01) via seamless cloning to obtain the recombinant plasmid pdCas12.

[0082] 2. Introduce the Cas12i_crRNA expression cassette into the editing vector.

[0083] A synthetically produced Cas12i_crRNA expression cassette, as shown in SEQ ID NO: 8, contains the rice U3 promoter shown in SEQ ID NO: 6, the nucleotide sequence encoding Cas12i3 DR shown in SEQ ID NO: 7, and a BsaI restriction enzyme site (GGTCTC). The Cas12i_crRNA expression cassette was amplified by PCR using primers crRNA-F / R. The nucleotide sequences of the primers are as follows:

[0084] crRNA-F: 5'-aaacgacggccagtgccaagcttaggaatctttaaacatacga-3';

[0085] crRNA-R: 5'-cgggtcacgctgcactgcagaaaaaacgagaccggcttgtccac-3'.

[0086] PCR system: KOD One™ PCR Master Mix (2x) 25 µL, crRNA-F (10 µM) 1.5 µL, crRNA-R (10 µM) 1.5 µL, synthesized Cas12i_crRNA expression cassette 1 µL, and PCR-grade pure water to a final volume of 50 µL. PCR program: 98°C pre-denaturation for 1-2 min; (98°C denaturation for 10 sec, 57°C annealing for 5 sec, 68°C extension for 5 sec) 30 cycles; final extension at 68°C for 2 min. After the reaction, the PCR products were detected by agarose gel electrophoresis, and the Cas12i_crRNA expression cassette fragment was recovered by gel excision.

[0087] The recombinant plasmid pdCas12 was digested with the restriction endonuclease KpnI (NEB, r3142). The digestion products were detected by agarose gel electrophoresis, and the pdCas12 linearized plasmid was recovered by gel excision. The Cas12i_crRNA expression cassette fragment was seamlessly ligated into the pdCas12 linearized plasmid using the ClonExpress II OneStep Cloning Kit (Novizan, C112-01) to obtain the recombinant plasmid pdCas12-crRNA.

[0088] 3. Introduce the target sequence into the editing vector

[0089] Target region: The promoter region of the rice genome IPA1 gene (Ideal Plant Architecture 1, LOC_Os08g39890). Sequence characteristics analysis of the IPA1 promoter region (2 kb upstream of the ATG) revealed a T-rich region (Cas12i target site, PAM: 5'-TTN-3', where N represents any nucleotide in the ATGC), a region unusable by the traditional Cas9 system. Based on methylation sequencing data from the literature (Zhang Lin et al., 2017), the DNA methylation variable region within the core regulatory region of the IPA1 promoter was selected as the target region.

[0090] To compare the ability of the CRISPR-dCas9 and CRISPR-dCas12i systems to regulate DNA methylation in the same region, target sequence 1 (SEQ ID NO: 9, located on the positive strand, upstream of the transcription start site, positions -584 to -559) was selected for constructing the OsSUVH2-dCas9 vector as a control; downstream of target sequence 1, target sequence 2 (SEQ ID NO: 10, located on the positive strand, upstream of the transcription start site, positions -562 to -540) was determined for constructing the CRISPR-dCas12i vector. The PAM sequence of target sequence 2 overlaps with the PAM sequence of target sequence 1 by 2 bases (…). Figure 2 ).

[0091] Target sequence 1: ccaccgtcgacgacagcgccgcttgg (SEQ ID NO: 9), PAM sequence TGG.

[0092] Target sequence 2: ttggcgctgccgccgtcttccgc (SEQ ID NO: 10), PAM sequence TTG.

[0093] The OsSUVH2-dCas9 vector was constructed using the method for preparing construct B1 disclosed in Example 1 of Chinese Invention Patent Application No. 202110046860.9. Except for a slight difference in the target sequence, the rest of the OsSUVH2-dCas9 vector is identical to construct B1 disclosed in that patent application. The primers used for annealing to form the gRNA coding sequence corresponding to target sequence 1 are T1-F and T1-R. The primers used for annealing to form the gRNA coding sequence corresponding to target sequence 2 are T2-F and T2-R. The nucleotide sequences of the primers are as follows:

[0094] T1-F: 5'-ggcgccaccgtcgacgacagcgccgct-3';

[0095] T1-R: 5'-aaacagcggcgctgtcgtcgacggtgg-3'.

[0096] T2-F: 5'-acacgcgctgccgccgtcttccgc-3';

[0097] T2-R: 5'-aaaagcggaagacggcggcagcgc-3'.

[0098] The annealing products formed after T2-F and T2-R annealing (DNA fragments encoding spacer sequences in gRNA with sticky ends) were ligated with the recombinant plasmid pdCas12-crRNA digested with restriction endonuclease BsaI (NEB, r3733) using T4 DNA ligase (NEB, m0202). This allowed the gRNA coding sequence corresponding to target sequence 2 (i.e., the fragments formed after T2-F and T2-R annealing) to be inserted into the BsaI restriction site of pdCas12-crRNA, resulting in the recombinant plasmid pdCas12-crRNAT2.

[0099] 4. Introducing the OsSUVH2 gene into the editing vector

[0100] Using cDNA from rice Zhonghua 11 (Oryza sativa subsp. japonica cv. Zhonghua 11) as a template, PCR was performed using primers OsSUVH2(AvrII)-F and OsSUVH2(AvrII)-R to amplify the OsSUVH2 gene, whose nucleotide sequence is shown in SEQ ID NO: 2. The nucleotide sequences of the primers are as follows:

[0101] OsSUVH2(AvrII)-F: 5'-gtttggtgttatacttctgcagcggtaccatggagatggacacatcgcc-3';

[0102] OsSUVH2(AvrII)-R: 5'-ctcgaccttcttcatcctaggcatggttaactttcccacc-3'.

[0103] PCR system: KOD One™ PCR Master Mix (2x) 25 µL, OsSUVH2(AvrII)-F (10 µM) 1.5 µL, OsSUVH2(AvrII)-R (10 µM) 1.5 µL, cDNA template 1 µL, PCR-grade pure water to a final volume of 50 µL. PCR program: 98°C pre-denaturation for 1-2 min; (98°C denaturation for 10 sec, 57°C annealing for 5 sec, 68°C extension for 10 sec) 30 cycles; 68°C final extension for 2 min. After the reaction, the PCR products were detected by agarose gel electrophoresis, and the OsSUVH2(AvrII) gene fragment was recovered by gel excision.

[0104] Using cDNA from rice Zhonghua 11 as a template, PCR was performed using primers OsSUVH2(SacI)-F and OsSUVH2(SacI)-R, following the PCR system and procedure described above, to amplify the OsSUVH2 gene, whose nucleotide sequence is shown in SEQ ID NO: 2. The nucleotide sequences of the primers are as follows:

[0105] OsSUVH2(SacI)-F: 5'-aggagtcaagcgcgtccggggagctcatggagatggacacatcgcc-3';

[0106] OsSUVH2(SacI)-R: 5'-gaacgaaagctctgagcttacatggttaactttcccaccc-3'.

[0107] After the reaction, the PCR products were detected by agarose gel electrophoresis, and the OsSUVH2(SacI) gene fragment was recovered by gel excision.

[0108] To compare the methylation modification efficiency, expression vectors (construction 1 and construct 2) were constructed to express fusion proteins with different topologies. Corresponding crRNA-free fusion protein expression vectors (construction 3 and construct 4) were also constructed as controls. No linker peptide coding sequences were found between the OsSUVH2 gene and the dCas12i-5M gene in constructs 1-4.

[0109] Construct 1 (Ubi-OsSUVH2-dCas12i3 + crRNA): The OsSUVH2 (AvrII) gene fragment was ligated to the pdCas12-crRNAT2 plasmid digested with the restriction endonuclease AvrII (NEB, r0174) using the ClonExpress II One Step Cloning Kit (Novizan, C112-01). The recombinant vector was obtained by seamlessly cloning the OsSUVH2 gene into the AvrII restriction site of the pdCas12-crRNAT2 plasmid. The vector expressed the fusion protein OsSUVH2-dCas12i3-5M, where dCas12i3-5M is located at the C-terminus of the fusion protein.

[0110] Construct 2 (Ubi-dCas12i3-OsSUVH2 + crRNA): The OsSUVH2 (SacI) gene fragment was ligated to the pdCas12-crRNAT2 plasmid digested with restriction endonuclease SacI (NEB, r3156) using the ClonExpress II One Step Cloning Kit (Novizan, C112-01). The OsSUVH2 gene was then inserted into the SacI restriction site of the pdCas12-crRNAT2 plasmid to obtain a recombinant vector that expresses the fusion protein dCas12i3-5M-OsSUVH2, where dCas12i3-5M is located at the N-terminus.

[0111] Construct 3 (Ubi-OsSUVH2-dCas12i3): The OsSUVH2 (AvrII) gene fragment was ligated to a pdCas12 plasmid digested with the restriction endonuclease AvrII (NEB, r0174) using the ClonExpress II One Step Cloning Kit (Novizan, C112-01). The OsSUVH2 gene was then seamlessly inserted into the AvrII restriction site of the pdCas12 plasmid to obtain a recombinant vector expressing the fusion protein OsSUVH2-dCas12i3-5M, where dCas12i3-5M is located at the C-terminus of the fusion protein. Construct 3 does not contain the Cas12i_crRNA expression cassette and serves as a control for Construct 1.

[0112] Construct 4 (Ubi-dCas12i3-OsSUVH2): The OsSUVH2 (SacI) gene fragment was ligated to a pdCas12 plasmid digested with the restriction endonuclease SacI (NEB, r3156) using the ClonExpress II One Step Cloning Kit (Novizan, C112-01). The OsSUVH2 gene was then seamlessly inserted into the SacI restriction site of the pdCas12 plasmid to obtain a recombinant vector expressing the fusion protein dCas12i3-5M-OsSUVH2, where dCas12i3-5M is located at the N-terminus. Construct 4 does not contain the Cas12i_crRNA expression cassette and serves as a control for Construct 2.

[0113] III. Validation of transient expression in rice protoplasts

[0114] Transient expression of the constructs in rice protoplasts was performed to verify their methylation regulatory activity. Following the method described in "Zhang, Yang, et al. "A highly efficient rice green tissue protoplast system for transient gene expression and studying light / chloroplast-related processes." Plant methods 7.1 (2011): 30.", protoplasts were extracted from etiolated seedlings of the rice variety Zhonghua 11 (ZH11) and transformed into constructs 1-4 and the OsSUVH2-dCas9 vector, respectively. After transformation, the protoplasts were cultured in the dark for 48 hours. Genomic DNA was extracted from the transformed protoplasts, and PCR was performed using hygromycin resistance gene-specific primers Hpt-F and Hpt-R to detect the hygromycin resistance gene (hyg), confirming that the constructs had been transformed into the protoplasts.

[0115] ‌Hpt-F: 5'-atgaaaaagcctgaactcaccgcgacgt-3';

[0116] ‌Hpt-R: 5'-ctatttctttgccctcggacgagt-3'.

[0117] Genomic DNA from protoplasts confirmed to be transformed into the construct was sulfite-treated using the Zymo Research EZ DNA Methylation-Lightning Kit (catalog number: D5030). Using the sulfite-treated genomic DNA as a template, a first round of PCR was performed using primers BSPSeq1-F1 and BSPSeq1-R1. A second round of PCR was performed using the first round PCR product as a template, using primers BSPSeq1-F2 and BSPSeq1-R2. The second round PCR product was purified and cloned into the TA cloning site of a T-vector (Promega, A3600) to obtain the recombinant vector. The recombinant vector was sequenced using universal primers to detect methylation levels near the target site.

[0118] BSPSeq1-F1:5'-ggttcgtcggagtagggg-3';

[0119] BSPSeq1-R1: 5'-atatcattaattatcttcttat-3'.

[0120] BSPSeq1-F2:5'-tagggcgttcggggagtttt-3';

[0121] BSPSeq1-R2: 5'-tttaacaaaatacaaaacaataa-3'.

[0122] The results are as follows Figure 3 As shown, construct 1 exhibits activity in increasing DNA methylation levels in the target region, but its activity is lower than that of the OsSUVH2-dCas9 vector; construct 2 does not exhibit activity in upregulating DNA methylation levels; constructs 3 and 4 do not have the function of regulating DNA methylation levels near the target site, indicating that this editing system has regulatory specificity. Therefore, the editing system will be further optimized using construct 1 as the base.

[0123] IV. Optimization of Constructs

[0124] 1. Introduction of linker peptides

[0125] To further improve the efficiency of DNA methylation regulation, a linker peptide was introduced between OsSUVH2 and dCas12i-5M, and the length and flexibility of the linker peptide were optimized. The following linker peptides were selected for experiments: 3×GGGGS linker peptide (SEQ ID NO: 11), 5×GGGGS linker peptide (SEQ ID NO: 12), XTEN linker peptide (SEQ ID NO: 13), 3×EAAAK linker peptide (SEQ ID NO: 14), 1×GGGGS-XTEN linker peptide (SEQ ID NO: 15), 2×GGGGS-XTEN linker peptide (SEQ ID NO: 16), and 3×GGGGS-XTEN linker peptide (SEQ ID NO: 17). GGGGS is a flexible linker peptide; XTEN is a semi-rigid linker peptide, which can greatly improve the solubility and stability of the fusion protein; and the 3×EAAAK linker peptide can form an α-helix structure, effectively isolating the two protein domains to prevent mutual interference.

[0126] The following is an example of artificially synthesized DNA encoding linker peptides with adapter sequences at both ends:

[0127] The nucleotide sequence encoding the 3×GGGGS linker peptide with a linker is: ggtgggaaagttaaccatgGGTGGCGGTGGCTCTGGTGGCGGTGGCTCTGGTGGCGGTGGCCTCTcctaggatgaagaaggtcgag;

[0128] The nucleotide sequence encoding the 5×GGGGS linker peptide with a linker is: ggtgggaaagttaaccatgGGTGGCGGTGGCTCTGGTGGCGGTGGCTCTGGTGGCGGTGGCTCTGGTGGCGGTGGCTCTGGTGGCGGTGGCTCTGGTGGCGGTGGCCTCTcctaggatgaagaaggtcgag;

[0129] The nucleotide sequence encoding the XTEN linker peptide with a linker is: ggtgggaaagttaaccatgAGCGGTAGCGAGACCCCGGGCACCAGCGAGAGCGCCACCCCGGAGAGCcctaggatgaagaaggtcgag;

[0130] Nucleotide sequence encoding 3×EAAAK linker peptide with linker: ggtgggaaagttaaccatgGAGGCCGCCGCCAAGGAGGCCGCCGCCAAGGAGGCCGCCGCCAAGcctaggatgaagaaggtcgag;

[0131] Nucleotide sequence encoding 1×GGGGS-XTEN linker peptide with linker: ggtgggaaagttaaccatgGGTGGCGGTGGCTCTAGCGGTAGCGAGACCCCGGGCACCAGCGAGAGCGCCACCCCGGAGAGCcctaggatgaagaaggtcgag;

[0132] Nucleotide sequence encoding 2×GGGGS-XTEN linker peptide with linker: ggtgggaaagttaaccatgGGTGGCGGTGGCTCTGGTGGCGGTGGCTCTAGCGGTAGCGAGACCCCGGGCACCAGCGAGAGCGCCACCCCGGAGAGCcctaggatgaagaaggtcgag;

[0133] The nucleotide sequence encoding the 3×GGGGS-XTEN linker peptide with a linker is: ggtgggaaagttaaccatgGGTGGCGGTGGCTCTGGTGGCGGTGGCTCTGGTGGCGGTGGCTCTAGCGGTAGCGAGACCCCGGGCACCAGCGAGAGCGCCACCCCGGAGAGCcctaggatgaagaaggtcgag.

[0134] PCR was performed using primers containing adapter sequences to amplify DNA encoding linker peptides at both ends. The nucleotide sequences of the primers are as follows:

[0135] Linker-F: 5'-ggtgggaaagttaaccatg-3';

[0136] Linker-R: 5'-ctcgaccttcttcatcctagg-3'.

[0137] PCR system: KOD One™ PCR Master Mix (2x) 25 µL, Linker-F (10 µM) 1.5 µL, Linker-R (10 µM) 1.5 µL, DNA template 1 µL, PCR-grade pure water to a final volume of 50 µL. PCR program: 98°C pre-denaturation for 1-2 min; (98°C denaturation for 10 sec, 57°C annealing for 5 sec, 68°C extension for 5 sec) 30 cycles; final extension at 68°C for 2 min. After the reaction, the PCR products were detected by agarose gel electrophoresis, and the DNA encoding linker peptides with adapter sequences at both ends was excised and recovered.

[0138] Construct 5: A recombinant vector was obtained by inserting DNA encoding the 3×GGGGS linker peptide with adapter sequences at both ends into the AvrII restriction site of Construct 1 (Ubi-OsSUVH2-dCas12i3 + crRNA), which expresses the fusion protein OsSUVH2-3×GGGGS-dCas12i3-5M.

[0139] Construct 6: A recombinant vector obtained by inserting DNA encoding the 5×GGGGS linker peptide with adapter sequences at both ends into the AvrII restriction site of Construct 1, which expresses the fusion protein OsSUVH2-5×GGGGS-dCas12i3-5M.

[0140] Construct 7: A recombinant vector obtained by inserting DNA encoding the XTEN linker peptide with adapter sequences at both ends into the AvrII restriction site of Construct 1, which expresses the fusion protein OsSUVH2-XTEN-dCas12i3-5M.

[0141] Construct 8: A recombinant vector obtained by inserting DNA encoding a 3×EAAAK linker peptide with adapter sequences at both ends into the AvrII restriction site of Construct 1, which expresses the fusion protein OsSUVH2-3×EAAAK-dCas12i3-5M.

[0142] Construct 9: A recombinant vector obtained by inserting DNA encoding the 1×GGGGS-XTEN linker peptide with adapter sequences at both ends into the AvrII restriction site of Construct 1, which expresses the fusion protein OsSUVH2-1×GGGGS-XTEN-dCas12i3-5M.

[0143] Construct 10: A recombinant vector obtained by inserting DNA encoding the 2×GGGGS-XTEN linker peptide with adapter sequences at both ends into the AvrII restriction site of Construct 1, which expresses the fusion protein OsSUVH2-2×GGGGS-XTEN-dCas12i3-5M.

[0144] Construct 11: A recombinant vector obtained by inserting DNA encoding the 3×GGGGS-XTEN linker peptide with adapter sequences at both ends into the AvrII restriction site of Construct 1, which expresses the fusion protein OsSUVH2-3×GGGGS-XTEN-dCas12i3-5M.

[0145] Constructs 5-11 were transiently expressed in etiolated protoplasts of the rice variety Zhonghua 11 (ZH11) to verify their methylation-regulating activity, following the same method described in Part III above. The results showed that construct 9 exhibited the best activity in regulating DNA methylation. Figure 4 and Figure 5The optimal effect is achieved when using a 1×GGGGS-XTEN linker peptide between OsSUVH2 and dCas12i-5M.

[0146] 2. Introduction of nuclear localization signal peptide

[0147] Because the fusion protein formed by OsSUVH2 and dCas12i-5M has a large molecular weight, it is difficult to enter the nucleus, which may affect its activity. To optimize activity, the SV40 nuclear localization signal peptide (SV40NLS) shown in SEQ ID NO: 18 and the Nucleoplasmin nuclear localization signal peptide (NucleoplasminNLS) shown in SEQ ID NO: 19 were introduced at both ends of the fusion protein OsSUVH2-1×GGGGS-XTEN-dCas12i3-5M. The coding sequences for SV40NLS shown in SEQ ID NO: 20 and NucleoplasminNLS shown in SEQ ID NO: 21 were artificially synthesized.

[0148] Construct 12 (SV-N_SV-C): A recombinant vector obtained by inserting the SV40NLS coding sequence into the KpnI and SacI restriction sites of Construct 9, respectively, expressing the fusion protein SV40NLS-OsSUVH2-1×GGGGS-XTEN-dCas12i3-5M-SV40NLS. SV40NLS is introduced at both ends of the fusion protein.

[0149] Construct 13 (Nu-N_Nu-C): A recombinant vector obtained by inserting the NucleoplasminNLS coding sequence into the KpnI and SacI restriction sites of Construct 9, respectively, expressing the fusion protein NucleoplasminNLS-OsSUVH2-1×GGGGS-XTEN-dCas12i3-5M-NucleoplasminNLS. NucleoplasminNLS is introduced at both ends of the fusion protein.

[0150] Construct 14 (SV-N_Nu-C): A recombinant vector obtained by inserting the SV40NLS coding sequence into the KpnI restriction site of Construct 9 and the NucleoplasminNLS coding sequence into the SacI restriction site of Construct 9, which expresses the fusion protein SV40NLS-OsSUVH2-1×GGGGS-XTEN-dCas12i3-5M-NucleoplasminNLS. SV40NLS is introduced at the N-terminus of the fusion protein, and NucleoplasminNLS is introduced at the C-terminus of the fusion protein.

[0151] Construct 15 (Nu-N_SV-C): A recombinant vector obtained by inserting the NucleoplasminNLS coding sequence into the KpnI restriction site of Construct 9 and the SV40NLS coding sequence into the SacI restriction site of Construct 9, which expresses the fusion protein NucleoplasminNLS-OsSUVH2-1×GGGGS-XTEN-dCas12i3-5M-SV40NLS. NucleoplasminNLS is introduced at the N-terminus of the fusion protein, and SV40NLS is introduced at the C-terminus of the fusion protein.

[0152] Constructs 12-15 were transiently expressed in etiolated protoplasts of the rice variety Zhonghua 11 (ZH11) to verify their methylation-regulating activity, following the same method described in Part III above. The results showed that construct 14 exhibited the best activity in regulating DNA methylation. Figure 6 The optimal combination is achieved when SV40NLS is located at the N-terminus and NucleoplasminNLS is located at the C-terminus (SV-N_Nu-C). The amino acid sequence of the fusion protein expressed by construct 14 is shown in SEQ ID NO: 22, and the nucleotide sequence encoding the fusion protein is shown in SEQ ID NO: 23. The nucleotide sequence encoding crRNA (i.e., guide RNA) in construct 14 is shown in SEQ ID NO: 25.

[0153] V. Acquisition of Genetically Modified Rice

[0154] The rice variety Zhonghua 11 (ZH11) was genetically transformed using construct 14, and the DNA methylation level of the transgenic plants was detected. The control group was transformed with construct 16 into rice ZH11. Construct 16 was prepared using construct 3 as the starting vector, following the construction method of construct 14. Construct 16 is identical to construct 14 except that it does not contain the Cas12i_crRNA expression cassette.

[0155] Constructs 14, 16, and the OsSUVH2-dCas9 vector were introduced into Agrobacterium EHA105 to obtain recombinant Agrobacterium. Following the rice genetic transformation method described in the reference "Hiei et al. (1994) (Hiei, Y., Ohta, S., Komari, T. and Kumashiro, T. (1994). Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. The Plant Journal, 6(2), 271-282.)," recombinant Agrobacterium was used to infect callus tissue of rice ZH11. After hygromycin selection, differentiation, and rooting, T0 generation transgenic regenerated plants were obtained. Genomic DNA was extracted from the leaves of the T0 generation plants and identified by PCR using the hygromycin resistance gene-specific primers Hpt-F and Hpt-R. The nucleotide sequences of the primers are described in Part III above. Transgenic positive plants could amplify a 1 kb fragment, while non-transgenic plants could not amplify this fragment. Gel electrophoresis results showed that transgenic positive plants containing construct 14, construct 16, and the OsSUVH2-dCas9 vector were obtained. Figure 7 ).

[0156] Five wild-type rice plants (ZH11), five transgenic T0 generation plants (construction 14, construct 16, and OsSUVH2-dCas9 vector) were collected for DNA methylation and IPA1 gene expression level detection. The method for DNA methylation level detection was the same as described in Part III above. RNA was extracted from wild-type rice plants (ZH11) and each transgenic T0 generation plant and reverse transcribed into cDNA. Using cDNA as a template, quantitative real-time PCR was performed using IPA1 gene-specific primers IPA1-F and IPA1-R. The OsActin1 gene (GenBank accession number: X16280) was used as an internal control (primers OsActin1-F / R) to detect IPA1 gene expression level. The nucleotide sequences of the primers are as follows:

[0157] IPA1-F: 5'-tgcattccaaggctccccgc-3';

[0158] IPA1-R: 5'-tgcggcagctgcgttttcct-3'.

[0159] OsActin1-F: 5'-cttcataggaatggaagctgcgggta-3';

[0160] OsActin1-R: 5'-cgaccaccttgatcttcatgctgcta-3'.

[0161] Quantitative real-time PCR was performed using SsoFast EvaGreen Supermixes (Bio-rad, catalog number: 1725201). PCR system: SsoFast EvaGreen Supermix (2x) 10 µL, forward primer (10 µM) 1.0 µL, reverse primer (10 µM) 1.0 µL, cDNA template 1.0 µL, Nuclease-free Water to a final volume of 20 µL. PCR program: Step 1: 95°C for 30 sec; Step 2: 95°C for 5 sec, 60°C for 10 sec, signal collection, 35 cycles; Step 3: Melting curve analysis, 65-95°C 0.5°C / step, 2 sec pause per step for signal collection. (Based on 2...) -ΔΔCt The relative expression level of IPA1 mRNA was calculated using this method.

[0162] DNA methylation level detection results are as follows: Figure 8 As shown, the methylation level of the T0 generation transgenic plants of construct 14 near target sequence 2 was significantly higher than that of the T0 generation transgenic plants of the OsSUVH2-dCas9 vector near target sequence 1 (Student's t-test, ***P<0.001). IPA1 gene expression levels are as follows... Figure 9 As shown, the IPA1 gene expression level in the T0 generation transgenic plants of construct 14 was significantly lower than that in wild-type rice ZH11 and OsSUVH2-dCas9 vector transgenic T0 generation plants (Student's t-test, ***P<0.001). The DNA methylation level and IPA1 gene expression level in the T0 generation transgenic plants of construct 16 showed no significant difference compared to wild-type rice ZH11. Figure 8 and Figure 9 ).

Claims

1. A fusion protein comprising, from N-terminus to C-terminus, a first nuclear localization signal peptide, a methylation-modified domain, a linker peptide, a nuclease-free Cas12i protein, and a second nuclear localization signal peptide; wherein the methylation-modified domain is an OsSUVH2 protein or a functionally active fragment thereof capable of upregulating methylation levels; the amino acid sequence of the OsSUVH2 protein is shown in SEQ ID NO: 1; the amino acid sequence of the nuclease-free Cas12i protein is shown in SEQ ID NO: 3; the amino acid sequence of the linker peptide is shown in SEQ ID NO: 15; the amino acid sequence of the first nuclear localization signal peptide is shown in SEQ ID NO: 18; and the amino acid sequence of the second nuclear localization signal peptide is shown in SEQ ID NO:

19.

2. The fusion protein according to claim 1, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID NO:

22.

3. Products used to increase the methylation level of specific regions of plant genomic DNA, comprising any one of the following (a)-(e): (a) The fusion protein of claim 1 or 2, and the guide RNA; (b) An expression construct 1 comprising a nucleotide sequence encoding the fusion protein of claim 1 or 2, and a guide RNA; (c) The fusion protein of claim 1 or 2, and the expression construct 2 comprising a nucleotide sequence encoding a guide RNA; (d) An expression construct 1 comprising a nucleotide sequence encoding the fusion protein of claim 1 or 2, and an expression construct 2 comprising a nucleotide sequence encoding a guide RNA; (e) An expression construct 3 comprising a nucleotide sequence encoding the fusion protein of claim 1 or 2 and a nucleotide sequence encoding a guide RNA; The guide RNA comprises: (i) spacer sequences capable of hybridizing with the target sequence in the specific region, and (ii) A direct repeat sequence attached to the spacer sequence; the direct repeat sequence is capable of guiding the fusion protein to bind to the guide RNA to form a CRISPR-Cas complex targeting the target sequence.

4. The product according to claim 3, characterized in that, In expression construct 1 and expression construct 3, the promoter used to initiate transcription of the nucleotide sequence encoding the fusion protein is the Ubi promoter; and / or In expression construct 2 and expression construct 3, the promoter used to initiate transcription of the nucleotide sequence encoding the guide RNA is the U3 promoter.

5. The product according to claim 3, characterized in that, The nucleotide sequence encoding the direct repeat sequence is shown in SEQ ID NO:

7.

6. The use of the product according to any one of claims 3-5 in improving the methylation level of specific regions of rice genomic DNA.

7. A method for increasing the methylation level of a specific region of rice genomic DNA, comprising: The product of any one of claims 3-5 is introduced into rice, wherein the specific region is the IPA1 gene promoter region, and the target sequence of the IPA1 gene promoter region is shown in SEQ ID NO: 10.

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