Gene down-regulation method based on regulation of precursor mRNA cleavage efficiency

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to a gene down-regulation method based on regulation of precursor mRNA (messenger Ribonucleic Acid) splicing efficiency, which comprises the following steps: firstly, identifying at least one intron 5'terminal splicing site containing a GT sequence in a target gene; aiming at the site, designing a guide RNA (Ribonucleic Acid) sequence capable of targeting the downstream fourth or fifth nucleotide, and verifying the cleavage activity of the guide RNA sequence; constructing a gene editing vector by using the guide RNA sequence which is verified to be effective and a Cas9 nuclease coding gene; introducing the vector into a plant cell, carrying out genetic transformation and regeneration to obtain a transformed plant, and screening out a mutant which is subjected to site-directed editing at a target site through DNA (Deoxyribose Nucleic Acid) sequencing; and finally, extracting RNA (Ribonucleic Acid) of the mutant, reversely transcribing into cDNA (Complementary Deoxyribonucleic Acid), and analyzing transcript change through PCR (Polymerase Chain Reaction) amplification and gel electrophoresis. According to the invention, stable, heritable and accurate knock-down of gene expression is realized, and a new tool is provided for essential gene function research and crop character improvement.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to a gene downregulation method based on regulating precursor mRNA cleavage efficiency. Background Technology

[0002] Outbreaks of plant diseases and pests not only reduce crop yields but also lower crop quality. Developing efficient and green agricultural control methods is crucial, and using disease-resistant varieties is the most economical, effective, and safe measure. Disease-resistant varieties can be developed not only through traditional hybridization breeding but also through transgenic breeding if superior resistance genes are discovered. Lesion-like mutants are plants that exhibit spontaneous necrosis caused by cell death in the absence of any pathogen infection or abiotic stress. Early studies have shown that many LMM plants exhibit stronger disease resistance compared to their ancestral parents, but their growth is inhibited.

[0003] However, these existing technologies generally share a common limitation. They primarily achieve "complete knockout" or "transient suppression" of gene function, making it difficult to achieve stable, heritable, and finely regulated "knockdown" of gene expression levels. Specifically, CRISPR-Cas9 knockout results in permanent loss of gene function, making it unsuitable for studying essential genes; while technologies such as CRISPRi, RNAi, and VIGS suffer from unstable effects, non-heritability, or high off-target risks, making them unsuitable for creating breeding materials with stably heritable and moderately reduced expression levels. Therefore, there is an urgent need in this field for a novel technological solution that can precisely, stably, and heritably downregulate gene expression, rather than completely knocking it out. Summary of the Invention

[0004] To overcome the above shortcomings, this invention provides a gene downregulation method based on regulating precursor mRNA cleavage efficiency, aiming to improve the problem that existing gene editing technologies are unable to achieve stable heritable gene downregulation with finely regulated expression levels in plants, rather than gene knockout.

[0005] In a first aspect, the present invention provides the following technical solution: a gene downregulation method based on regulating precursor mRNA cleavage efficiency, the method comprising the following steps: S1. Identify the 5'-GT splicing site in the target gene: In the DNA sequence of the target gene, identify the 5' splicing site of at least one intron, the 5' splicing site containing the GT dinucleotide sequence; S2. Design and Validation of Guide RNA: Design at least one guide RNA sequence that can target the 4th or 5th nucleotide downstream of the 5'-GT splicing site identified in S1, and verify that the guide RNA sequence has specific cleavage activity against the DNA fragment containing the site in a system containing Cas9 nuclease. S3. Constructing a gene editing vector: The guide RNA sequence verified in S2 is cloned into an expression vector containing the Cas9 nuclease coding sequence to construct a gene editing vector; S4. Genetic transformation and screening of mutants: The gene editing vector constructed in S3 is introduced into the target plant cells or tissues, and a transformed plant population is obtained through genetic transformation and regeneration. From the transformed plant population, mutant plants that have undergone site-directed editing at the 4th or 5th nucleotide downstream of the 5'-GT splice site are screened by DNA sequencing. S5. Transcript analysis: RNA was extracted from the mutant plants obtained in S4 and cDNA was obtained by reverse transcription; S6. Detecting transcript changes: Using the cDNA obtained in S5 as a template, PCR amplification is performed using primers that cross the editing site, and the amplification products are subjected to gel electrophoresis to obtain the transcript electrophoresis pattern of the target gene.

[0006] Preferably, in S1, the process of identifying the 5' splice site of at least one intron includes: Obtain the full-length DNA sequence of the target gene; In the full-length DNA sequence, the 5' boundaries of all introns were scanned and located one by one; Extract and record the nucleotide sequence starting at the 5' boundary of each intron, the sequence containing GT dinucleotides; From the recorded nucleotide sequences, 5' splicing sites containing conserved GUAAG or GTAA sequences were screened out.

[0007] Preferably, in S2, the process of designing at least one guide RNA sequence capable of targeting its downstream 4th or 5th nucleotide includes: Based on the nucleotide sequence of the selected 5' splice site, the positions of the 4th and 5th nucleotides downstream of it are determined; The target sequence is a sequence of at least 20 consecutive nucleotides containing the 4th or 5th nucleotide. The target sequence is linked to the guide RNA backbone sequence of the Cas9 nuclease to form a guide RNA sequence.

[0008] Preferably, in S2, the process of verifying that the guide RNA sequence has specific cleavage activity against the DNA fragment containing the site in a system containing Cas9 nuclease includes: Construct a test expression vector containing the guide RNA sequence and the Cas9 nuclease encoding gene; The test expression vector was introduced into plant protoplasts for transient expression. Genomic DNA was extracted from the plant protoplasts and amplified by PCR using primers that span the 5'-GT splice site. If the PCR amplification product is detected and a smaller DNA fragment than the control is found, the guide RNA sequence is confirmed to have specific cleavage activity.

[0009] Preferably, in step S3, the process of constructing the gene-editing vector includes: The constructed guide RNA sequence is inserted into a plant expression vector through an enzyme digestion and ligation reaction to form an intermediate vector containing a single guide RNA sequence expression cassette. The Cas9 nuclease coding sequence is inserted into a specific position in the intermediate vector through an enzyme digestion and ligation reaction, so that the guide RNA sequence expression cassette and the Cas9 nuclease coding sequence are in the same expression frame or a separate expression frame, thus constructing the gene editing vector.

[0010] Preferably, in step S4, the process of introducing the gene-editing vector constructed in step S3 into target plant cells or tissues, and obtaining a transformed plant population through genetic transformation and regeneration, includes: The constructed gene-editing vector was transformed into Agrobacterium competent cells to obtain engineered Agrobacterium; The engineered Agrobacterium was used to infect explants of the target plant; The infected explants were co-cultured and screened on a culture medium containing screening antibiotics to induce callus formation. The callus tissue was cultured on differentiation and rooting medium to regenerate complete transformed plants.

[0011] Preferably, in S4, the process of screening mutant plants from the transformed plant population by DNA sequencing for site-directed editing at the 4th or 5th nucleotide downstream of the 5'-GT splice site includes: Genomic DNA was extracted from the transformed plants; Using the genomic DNA as a template, PCR amplification was performed using the primer pairs shown in SEQ ID NO: 1 and SEQ ID NO: 2 to obtain a DNA fragment containing the 5'-GT splice site; The PCR amplification product was sequenced to obtain the nucleotide sequence of the 5'-GT splice site region; The obtained nucleotide sequence is compared with the unedited wild-type sequence. If the nucleotide at position 4 or 5 downstream of the 5'-GT splice site is deleted, inserted, or replaced, the plant is determined to be a site-edited mutant plant.

[0012] Preferably, in step S5, the process of extracting RNA from the mutant plant obtained in step S4 and reverse transcribing it to obtain cDNA includes: Tissues from the mutant plants identified as having undergone site-specific editing were collected, and total RNA was extracted using an RNA extraction kit. The concentration and purity of the extracted total RNA were determined; Using qualified total RNA as a template, reverse transcription was performed using reverse transcriptase and oligo(dT) or random primers to synthesize first-strand cDNA.

[0013] Preferably, in step S6, the process of performing PCR amplification using the cDNA obtained in step S5 as a template and primers spanning the edited site includes: Using the synthesized cDNA as a template, a primer pair consisting of the upstream primer shown in SEQ ID NO: 1 and the downstream primer shown in SEQ ID NO: 2 was used; PCR amplification was performed in a reaction system containing DNA polymerase, dNTPs, and magnesium ions.

[0014] Preferably, in step S6, the procedure for performing gel electrophoresis on the amplification products to obtain the transcript electrophoretic pattern of the target gene includes: Mix the obtained PCR amplification products with DNA loading buffer; The mixed sample was loaded into the wells of the agarose gel, and DNA molecular weight standards were loaded at the same time. Electrophoresis was performed at a constant voltage until the bromophenol blue indicator migrated to the vicinity of the bottom of the gel. The gel after electrophoresis was stained in nucleic acid staining solution and imaged under ultraviolet or blue light to obtain the transcript electrophoresis pattern showing DNA bands of different sizes.

[0015] The present invention has the following beneficial effects: 1. In this invention, the splicing efficiency of precursor mRNA is precisely disrupted by single-base editing, and the abundance of functional transcripts is reduced by the cell’s own nonsense-mediated degradation pathway. This creates a series of sub-effective allele materials with different degrees of reduced expression, and this trait can be stably inherited by offspring, overcoming the limitations of traditional CRISPR knockout and RNAi / VIGS.

[0016] 2. In this invention, the expression of disease resistance can be precisely downregulated without significantly affecting the normal growth and fruit setting of plants, thereby balancing the long-standing contradiction between disease resistance and agronomic traits, and providing a brand-new technical path for crop disease resistance breeding.

[0017] 3. In this invention, minimally invasive genome editing is achieved by targeting highly conserved RNA splicing core sequences with low off-target risk. At the same time, since it targets a conserved splicing mechanism, this technology is expected to have wide applicability in a variety of plants and even other organisms, providing a universal solution for solving similar problems in different species. Attached Figure Description

[0018] Figure 1 This is a schematic diagram illustrating the principle of the gene downregulation technique based on regulating precursor mRNA cleavage efficiency proposed in this invention. Figure 2 This is a schematic diagram of the conserved splicing site of OsRBL1 in the gene downregulation method based on regulating precursor mRNA splicing efficiency proposed in this invention. Figure 3 This is a schematic diagram of the detection of OsRBL1 intron three- and four-point mutation transcripts based on the gene downregulation method for regulating precursor mRNA splicing efficiency proposed in this invention. Figure 4 This is a schematic diagram of the single-target Cas9NG1 and Cas9NG3 vectors for the gene downregulation method based on regulating precursor mRNA cleavage efficiency proposed in this invention. Figure 5 This is a schematic diagram of the dual-target Cas9NG1NG3 and Cas9NG2NG3 vectors for the gene downregulation method based on regulating precursor mRNA cleavage efficiency proposed in this invention. Figure 6 This is a schematic diagram of the dual-target Cas9NG1NG3 target cleavage of the gene downregulation method based on regulating precursor mRNA cleavage efficiency proposed in this invention. Figure 7 This is a schematic diagram of the base pairs of the mutant rbl1^del^ and rbl1^ins^ mutation sites in the gene downregulation method based on regulating precursor mRNA cleavage efficiency proposed in this invention. Figure 8 This is a schematic diagram of the growth of mutants rbl1^del^ and rbl1^ins^ and transcript detection gel based on the gene downregulation method for regulating precursor mRNA cleavage efficiency proposed in this invention. Detailed Implementation

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] In a first embodiment of the present invention, the present invention provides a gene downregulation method based on regulating precursor mRNA cleavage efficiency, such as... Figures 1-8 As shown, it includes the following steps: S1. Identify the 5'-GT splicing site in the target gene: In the DNA sequence of the target gene, identify the 5' splicing site of at least one intron, which contains the GT dinucleotide sequence.

[0021] Furthermore, in S1, the procedure for identifying the 5' splice site of at least one intron includes: Obtain the full-length DNA sequence of the target gene; In the full-length DNA sequence, scan and locate the 5' boundaries of all introns one by one; Extract and record the nucleotide sequence starting at the 5' boundary of each intron, the sequence containing GT dinucleotides; From the recorded nucleotide sequences, 5' splicing sites containing conserved GUAAG or GTAA sequences were screened out.

[0022] Specifically, firstly, the full-length DNA sequence of the target gene is obtained. This sequence can be obtained by querying public genome databases or by de novo sequencing using molecular cloning and sequencing technologies. Secondly, bioinformatics analysis is performed on the obtained full-length DNA sequence. Using sequence analysis software or a script, the 5' boundaries of all introns are scanned and located one by one in the DNA sequence. The 5' boundary of an intron is the junction between it and an exon. A predetermined length of nucleotide sequence is extracted and recorded for each located intron, starting at its 5' boundary. For example, 10 to 20 nucleotides are extracted extending into the intron from the aforementioned boundary. Each extracted sequence must contain the GT dinucleotide located at the boundary, i.e., the sequence must begin with GT. Based on the known conserved sequence characteristics of splicing sites, all recorded nucleotide sequences are screened. Specifically, the sequence immediately following the GT dinucleotide in each sequence is examined. If the sequence contains the highly conserved nucleotide motif GUAAG or GTAA, the corresponding intron 5' boundary is determined to be a candidate 5'-GT splicing site that meets the screening criteria of this invention. For example, in the target gene OsRBL1, this process successfully identified multiple 5'-GT splicing sites containing such conserved sequences, such as the third and fourth introns. Finally, the sequence information and location coordinates of one or more candidate 5'-GT splicing sites were recorded and output for use in the design of guide RNA in subsequent steps.

[0023] S2. Design and Validation of Guide RNA: Design at least one guide RNA sequence that can target the 4th or 5th nucleotide downstream of the 5'-GT splicing site identified in S1, and verify that the guide RNA sequence has specific cleavage activity against the DNA fragment containing the site in a system containing Cas9 nuclease.

[0024] Furthermore, in S2, the process of designing at least one guide RNA sequence capable of targeting its downstream 4th or 5th nucleotide includes: Based on the nucleotide sequence of the selected 5' splice site, the positions of the 4th and 5th nucleotides downstream of it were determined; The target sequence is a continuous sequence of at least 20 nucleotides containing the 4th or 5th nucleotide. The target sequence is linked to the guide RNA backbone sequence of the Cas9 nuclease to form the guide RNA sequence.

[0025] Furthermore, in S2, the procedure for verifying that the guide RNA sequence has specific cleavage activity against DNA fragments containing the Cas9 nuclease in a system containing the Cas9 nuclease includes: Construct a test expression vector containing a guide RNA sequence and the Cas9 nuclease encoding gene; The test expression vector was introduced into plant protoplasts for transient expression. Genomic DNA was extracted from plant protoplasts and amplified by PCR using primers that span the 5'-GT splice site. If the PCR amplification products are detected and smaller DNA fragments are found compared to the control, it confirms that the guide RNA sequence has specific cleavage activity.

[0026] Specifically, based on the nucleotide sequences of candidate 5' splicing sites selected in the above steps, the precise location of the site is first determined. The position of the first base G of the GT dinucleotide is designated as +1, and the immediately downstream bases are designated as +2 and +3. The key target positions of this invention are the +4 and +5 nucleotides downstream of this site. At least 20 consecutive nucleotide sequences, including the aforementioned +4 or +5 nucleotides, are used as the target sequence for the guide RNA. The design of this target sequence must adhere to the general requirements of the CRISPR-Cas9 system for guide RNA target sequences, such as avoiding multi-T structures and assessing potential off-target effects. For example, a corresponding target sequence can be designed for a specific splicing site in the OsRBL1 gene. Then, the DNA fragment containing the target sequence is obtained through chemical synthesis or PCR amplification. Using molecular cloning techniques, this target sequence is precisely inserted into an expression cassette containing the universal guide RNA backbone sequence of the Cas9 system. Typically, the target sequence is cloned to a specific site upstream of the backbone sequence, so that after transcription in the vector, the target sequence and the backbone sequence together form a complete guide RNA transcript with secondary structure. This step ultimately constructs a DNA module containing a customized guide RNA coding sequence. To verify whether the designed guide RNA can guide the Cas9 nuclease to specifically cleave at the target site, in vitro or in vivo cleavage activity verification is required. First, a test expression vector is constructed. The constructed DNA module containing the customized guide RNA coding sequence is co-cloned with the Cas9 nuclease coding gene into a vector backbone suitable for transient expression in plant cells. This vector should contain a promoter that functions in plant cells to drive the expression of the Cas9 gene and the guide RNA. For example, a binary vector simultaneously expressing the Cas9 protein and a specific guide RNA can be constructed. Second, transient transformation and expression in plant protoplasts are performed. Actively growing plant seedlings are selected, and protoplasts are prepared using enzymatic digestion. The constructed test expression vector is introduced into the protoplasts using a polyethylene glycol-mediated transformation method. The transformed protoplasts are cultured under suitable conditions for a specific time, such as 16 to 24 hours, to allow the exogenous Cas9 protein and guide RNA to be expressed and perform cleavage function. Then, genomic DNA is extracted and PCR detection is performed. The cultured protoplasts are collected, and total genomic DNA is extracted using a plant genomic DNA extraction kit. Using this genomic DNA as a template, a PCR reaction was performed using a pair of primers specifically designed to amplify the region containing the target 5'-GT splice site. The primer pair should be designed to ensure that the amplification product covers the regions flanking the expected cleavage site of the guide RNA. PCR amplification products were separated by agarose gel electrophoresis, stained with nucleic acid dyes, and observed under UV light. A control experiment was also set up, for example, protoplasts transformed with an empty vector or a Cas9 vector without a guide RNA expression cassette served as a negative control. If the electrophoretic pattern of the PCR products in the experimental group showed one or more smaller, specific bands below the expected full-length band, while the negative control group showed only a single full-length band, it demonstrated that the designed guide RNA successfully guided Cas9 to specifically cleave at the target site, indicating that the guide RNA sequence was active. For example, when validating a guide RNA targeting the OsRBL1 gene, this method was used to observe the expected size of the fragment deletion, confirming cleavage activity.

[0027] S3. Constructing a gene editing vector: The validated guide RNA sequence from S2 is cloned into an expression vector containing the Cas9 nuclease coding sequence to construct a gene editing vector.

[0028] Furthermore, in S3, the process of constructing a gene-editing vector includes: The constructed guide RNA sequence is inserted into a plant expression vector through an enzyme digestion and ligation reaction to form an intermediate vector containing a single guide RNA sequence expression cassette. The Cas9 nuclease coding sequence is inserted into a specific position in an intermediate vector through an enzyme digestion and ligation reaction, so that the guide RNA sequence expression cassette and the Cas9 nuclease coding sequence are in the same expression frame or a separate expression frame, thus constructing a gene editing vector.

[0029] Specifically, a guide RNA sequence with verified specific cleavage activity was constructed into a plant expression vector suitable for stable transformation. Restriction endonucleases were used to digest the DNA fragment containing the guide RNA coding sequence and the selected plant binary expression vector, respectively, to generate compatible ends. The digested guide RNA fragment was then directionally inserted into the multiple cloning site of the vector using DNA ligase. This site is typically located below a promoter suitable for expression in plant cells. The ligation product was transformed into competent *E. coli* cells, and positive clones containing the correct inserted fragment were obtained through antibiotic selection and colony PCR verification. This resulted in the construction of an intermediate vector containing a complete single guide RNA expression cassette driven by a plant promoter. Subsequently, the coding sequence of the Cas9 nuclease is integrated into the aforementioned intermediate vector. The coding gene for the Cas9 nuclease can be derived from *Streptococcus pyogenes*, and plant codon optimization can be performed as needed. Restriction endonucleases are used to digest the donor plasmid containing the Cas9 coding gene and the aforementioned constructed intermediate vector to release the Cas9 gene fragment and open a specific insertion site on the intermediate vector. The selection of this insertion site must ensure that the Cas9 gene can be effectively expressed. The Cas9 gene fragment is then ligated to the linearized intermediate vector using a ligase. Two main expression strategies are available during this process. The first strategy is to construct a separate expression cassette, where the Cas9 gene is inserted after a separate plant promoter on the vector, allowing the guide RNA and Cas9 protein to be expressed by two separate transcription units. The second strategy is to construct the same expression cassette, for example, by using a 2A self-cleaving peptide sequence to link the Cas9 gene and the guide RNA expression cassette in the same transcript, resulting in separate Cas9 protein and guide RNA after translation. The ligation product was transformed into *E. coli* again, screened using a medium containing the appropriate antibiotics, and plasmids were extracted for restriction enzyme mapping analysis and sequencing verification to ensure that the Cas9 gene and guide RNA expression cassette were correctly integrated into the vector and expressed in the correct direction. This ultimately yielded a complete gene editing vector suitable for stable plant genetic transformation, such as Cas9-NG1 or Cas9-NG3 vectors for targeted editing of the OsRBL1 gene.

[0030] S4. Genetic transformation and screening of mutants: The gene editing vector constructed in S3 is introduced into the target plant cells or tissues, and a transformed plant population is obtained through genetic transformation and regeneration. From the transformed plant population, mutant plants with site-directed editing at the 4th or 5th nucleotide downstream of the 5'-GT splice site are screened by DNA sequencing.

[0031] Furthermore, in S4, the process of introducing the gene-editing vector constructed in S3 into target plant cells or tissues, and obtaining a transformed plant population through genetic transformation and regeneration includes: The constructed gene-editing vector was transformed into Agrobacterium competent cells to obtain engineered Agrobacterium; Using engineered Agrobacterium to infect explants of target plants; Infected explants were co-cultured and screened on a culture medium containing screening antibiotics to induce callus formation. The callus tissue was cultured on differentiation and rooting medium to regenerate complete transformed plants.

[0032] Furthermore, in S4, the procedure for screening mutant plants from the transformed plant population by DNA sequencing to identify those with site-directed editing at the 4th or 5th nucleotide downstream of the 5'-GT splice site includes: Genomic DNA was extracted from the transformed plants; Using genomic DNA as a template, PCR amplification was performed using primer pairs as shown in SEQ ID NO: 1 and SEQ ID NO: 2 to obtain DNA fragments containing 5'-GT splicing sites; The PCR amplification products were sequenced to obtain the nucleotide sequence of the 5'-GT splice site region; The obtained nucleotide sequence is compared with the unedited wild-type sequence. If the nucleotide at position 4 or 5 downstream of the 5'-GT splice site is deleted, inserted, or replaced, the plant is determined to be a site-edited mutant plant.

[0033] Specifically, the constructed gene-editing vector is introduced into Agrobacterium competent cells via electroporation or freeze-thaw methods, such as Agrobacterium tumefaciens strains EHA105 or GV3101. The transformation mixture is spread on solid LB medium containing the appropriate antibiotics and cultured at a suitable temperature until single colonies appear. Single colonies are picked for colony PCR verification to amplify the specific fragments on the vector to confirm successful transformation into Agrobacterium. Positive clones that have been verified are expanded, and the cells are collected and resuspended in infection medium to prepare an engineered Agrobacterium suspension. Healthy target plant seeds are selected, surface-sterilized, and germinated under aseptic conditions. Specific tissues of the germinated seedlings are used as explants, such as mature or immature embryos of rice. The engineered Agrobacterium suspension is used to immerse and infect the explants, with brief vacuum aspiration during this process to promote Agrobacterium attachment. After infection, the explants are transferred to co-culture medium and cultured in the dark at a suitable temperature for several days, allowing Agrobacterium to transfer the T-DNA region, containing the gene-editing vector, into the plant cells. After co-culture, explants were transferred to a sterile selection medium containing selection antibiotics and antibacterial agents. The selection antibiotics killed untransformed plant cells, allowing only those cells integrating the plant selection marker genes from the vector to survive and proliferate. After several weeks of culture on this medium, resistant callus formation was induced in the explants. The growing callus was periodically transferred to fresh selection medium for subculture to maintain selection pressure and promote callus expansion. Vigorously growing resistant callus was then transferred to differentiation medium. Cultured under light conditions, callus differentiation into green shoots was induced. Once the green shoots reached a certain size, they were cut and transferred to rooting medium to induce root development. Finally, the rooted, intact plantlets were transplanted into a sterile substrate and cultured under controlled conditions to obtain a population of complete transformed plants regenerated from transformed cells, i.e., the T0 generation plants. Young leaf tissues were extracted from the T0 generation transformed plants obtained above, and total genomic DNA was extracted using a plant genomic DNA extraction kit. The extracted genomic DNA was used as a template for PCR amplification. A pair of specific primers was used for the PCR reaction, with nucleotide sequences SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The sequence of SEQ ID NO: 1 is 5'-GCACTTCTTTTTCACAGCAA-3', and the sequence of SEQ ID NO: 2 is 5'-ATCATGTGCGTCCAGGCATA-3'. This primer pair was designed to amplify DNA fragments containing the target 5'-GT splice site and its upstream and downstream sequences. Configure the PCR reaction system and perform amplification. The amplification program includes pre-denaturation, cyclic amplification, and final extension. The annealing temperature for cyclic amplification is set to 60 degrees Celsius, and the number of cycles is 35. After amplification, a portion of the PCR product is subjected to agarose gel electrophoresis to confirm the specific amplification band of the expected size. The remaining PCR product is purified and then sent to a sequencing company. One of the PCR primers is used as the sequencing primer to perform Sanger DNA sequencing on the purified product. A clear sequencing map containing the target 5'-GT splice site region is obtained. The nucleotide sequence obtained from the sequencing is compared with the corresponding sequence obtained from wild-type non-transgenic plants using the same method. The comparison focuses on the sequences at the 4th and 5th nucleotides downstream of the 5'-GT splice site. If one or more bases are deleted, an additional base is inserted, or a base substitution is found at this location, the T0 generation plant is determined to be a mutant plant with site-directed editing at the target site. For example, a homozygous mutant rbl1^del^ with a deletion of the fifth base of intron 3 in the OsRBL1 gene and a homozygous mutant rbl1^ins^ with an insertion of adenine at the fourth base of intron 4 were obtained through screening.

[0034] S5. Transcript analysis: RNA was extracted from the mutant plants obtained in S4 and cDNA was obtained by reverse transcription.

[0035] Furthermore, in S5, the process of extracting RNA from the mutant plants obtained in S4 and reverse transcribing it to obtain cDNA includes: Tissues from mutant plants identified as having undergone site-directed editing were collected, and total RNA was extracted using an RNA extraction kit. The concentration and purity of the extracted total RNA were determined; Using qualified total RNA as a template, reverse transcription was performed using reverse transcriptase and oligo(dT) or random primers to synthesize first-strand cDNA.

[0036] Specifically, firstly, mutant plants verified by sequencing as site-directed editing were selected. Simultaneously, wild-type plants of the same species that had not undergone genetic transformation were used as controls. Fresh, young tissues, such as leaves or young shoots, were collected from the plants and rapidly frozen in liquid nitrogen to prevent RNA degradation. Total RNA was extracted using a plant total RNA extraction kit. The frozen tissue samples were thoroughly ground into a fine powder in liquid nitrogen, and then the kit was followed according to the instructions. The main steps included thoroughly lysing cells with lysis buffer, removing cell debris and genomic DNA by centrifugation, specifically adsorbing RNA onto a silica membrane column, removing impurities with washing buffer, and finally eluting high-purity total RNA from the membrane with RNase-free elution buffer. The concentration of extracted total RNA was determined using a micro-spectrophotometer. Simultaneously, the purity of the total RNA was assessed by measuring the absorbance ratio at 260 nm and 280 nm wavelengths. Generally, a ratio between 1.8 and 2.1 indicates minimal contamination from proteins and other impurities. Furthermore, the integrity of the total RNA was checked by agarose gel electrophoresis. Clear 28S and 18S rRNA bands without significant degradation tails indicated acceptable RNA quality. Using this high-quality total RNA as a template, reverse transcription was performed to synthesize complementary DNA. The reaction system was prepared in sterile, RNase-free centrifuge tubes using a commercially available reverse transcription kit. The reaction system contained a specific amount of total RNA template, oligo(dT) primers or random hexamer primers, a mixture of dNTPs, reverse transcriptase, RNase inhibitors, and the corresponding reaction buffer. If oligo(dT) primers are used, they specifically bind to the 3' poly(A) tail of eukaryotic mRNA, making them suitable for reverse transcription from mature mRNA containing poly(A) tails. If random primers are used, they bind randomly to the entire RNA population, making them suitable for reverse transcription of all RNA, including precursor mRNA, and particularly useful for detecting transcripts that may have splicing abnormalities. After mixing the reaction mixture, place it in a PCR instrument and perform the reaction according to the kit's recommended procedure. The reaction procedure typically includes a primer annealing period, a reverse transcriptase extension period, and a final enzyme inactivation step. After the reaction, a cDNA product containing the target gene transcript information is obtained. This cDNA product can be used directly for subsequent PCR amplification or stored at -20°C for later use. Using this procedure, cDNA for transcript analysis was successfully obtained from rbl1^del^ and rbl1^ins^ mutants and their wild-type control plants.

[0037] S6. Detecting transcript changes: Using the cDNA obtained in S5 as a template, PCR amplification was performed using primers that cross the editing site, and the amplification products were subjected to gel electrophoresis to obtain the transcript electrophoresis pattern of the target gene.

[0038] Furthermore, in S6, the procedure for PCR amplification using the cDNA obtained in S5 as a template and primers spanning the editing site includes: Using the synthesized cDNA as a template, a primer pair consisting of the upstream primer shown in SEQ ID NO: 1 and the downstream primer shown in SEQ ID NO: 2 was used; PCR amplification was performed in a reaction system containing DNA polymerase, dNTPs, and magnesium ions.

[0039] Furthermore, in S6, the procedure for performing gel electrophoresis on the amplification products to obtain the transcript electrophoresis pattern of the target gene includes: Mix the obtained PCR amplification products with DNA loading buffer; The mixed sample was loaded into the wells of the agarose gel, and DNA molecular weight standards were loaded at the same time. Electrophoresis was performed at a constant voltage until the bromophenol blue indicator migrated to the vicinity of the bottom of the gel. After electrophoresis, the gel is stained in nucleic acid staining solution and imaged under ultraviolet or blue light to obtain transcript electrophoresis patterns showing DNA bands of different sizes.

[0040] Specifically, firstly, polymerase chain reaction (PCR) amplification is performed using the synthesized cDNA product as a template. The primer pair used in the PCR reaction is the same as the primer pair used for genomic DNA screening, with nucleotide sequences SEQ ID NO: 1 and SEQ ID NO: 2, respectively. SEQ ID NO: 1 serves as the upstream primer, and SEQ ID NO: 2 serves as the downstream primer. This primer pair is designed in the exon region of the target gene and can cross the introns where the edited splice sites are located, thereby amplifying all transcript cDNA products containing information on normal and abnormal splicing. Prepare the PCR reaction system. The system contains the following components: a certain volume of template cDNA, for example, 1 μL; 2 μL each of SEQ ID NO: 1 primer and SEQ ID NO: 2 primer at a concentration of 10 μmol; 25 μL of high-fidelity DNA polymerase premix, which already contains DNA polymerase, dNTPs, magnesium ions, and reaction buffer; and finally, add nuclease-free water to bring the total volume to 50 μL. After thoroughly mixing the reaction system, place it in a thermal cycler and set and execute the PCR amplification program. The program includes: an initial pre-denaturation step, held at 98°C for 2 minutes; then 35 cycles of amplification, each cycle including denaturation at 98°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 1 minute; after the cycles, a final extension step is performed, held at 72°C for 5 minutes. After the reaction, the PCR amplification product is obtained. Take an appropriate amount of PCR amplification product and mix it with 6x DNA loading buffer containing indicator dye at a volume ratio of 5:1 to prepare a 1.5% agarose gel. Weigh an appropriate amount of agarose powder, add 1x TAE buffer, heat to dissolve, cool to approximately 60°C, add nucleic acid dye and mix well, pour into a gel casting tank, insert a comb with an appropriate number of wells, and allow to solidify. Place the solidified agarose gel into an electrophoresis tank containing 1x TAE electrophoresis buffer and remove the comb. Add the PCR product sample mixed with loading buffer to the loading wells of the gel. At the same time, add a DNA molecular weight standard with a known molecular weight range, such as DL2000 DNA Marker, to one loading well. Turn on the power, set a constant voltage of 120 volts, and start electrophoresis. Observe the migration position of the indicator dye bromophenol blue. Stop electrophoresis when the bromophenol blue band migrates to about three-quarters to the full length of the gel at the bottom. Remove the gel from the electrophoresis tank. If the nucleic acid dye used is not pre-prepared, place the gel in a staining solution containing ethidium bromide or other alternative dyes and stain at room temperature for 15 to 20 minutes. After staining, destain the gel appropriately with deionized water and place the stained gel on the sample stage of the gel imaging system. Image the gel under ultraviolet or blue light excitation to obtain an electrophoretic pattern clearly showing DNA bands. Analyze transcript changes by comparing the banding patterns of mutant plants with wild-type control plants. For example, in the rbl1^del^ and rbl1^ins^ mutants of the OsRBL1 gene, the electrophoretic pattern shows multiple bands or bands with different migration positions compared to the single band in the wild type, indicating abnormal transcript splicing and a decrease in the proportion of normal transcripts.

[0041] SEQ ID NO: 1: An oligonucleotide sequence with the sequence: 5'-GCACTTCTTTTTCACAGCAA-3'. This sequence is used as the upstream primer for PCR amplification in this paper; SEQ ID NO: 2: An oligonucleotide sequence with the sequence: 5'-ATCATGTGCGTCCAGGCATA-3'. This sequence is used as a downstream primer for PCR amplification in this paper. The method of this invention has broad applicability. Its core lies in targeting the highly conserved 5'-GT splicing site within the intron of a gene and its specific downstream position (position 4 or 5). The following are examples of target genes that have been experimentally verified or have application potential, to illustrate the feasibility and universality of this technology, but should not be construed as limiting the invention: 1. Core Implementation Gene: OsRBL1, a rice (Oryza sativa) lesion mutant gene. Its genome sequence is well-known in the art and can be obtained from public databases. This invention has successfully created downregulated mutants by targeting specific splicing sites in its third and fourth introns; 2. Key detection primer sequences: primer pairs used to amplify the target region of the OsRBL1 gene, the nucleotide sequences of which are shown in SEQ ID NO: 1 and SEQ ID NO: 2 as described in this specification; 3. Examples of other target genes for scalable applications (not exhaustive): Genes related to other plant lesion mutants, such as the OsCul3a and OsSPL28 genes in rice, and the ZmLLs1 and ZmLes22 genes in maize. These genes all contain conserved splicing sites that can be targeted by the method of this invention; Essential plant genes or lethal mutation genes, such as the centromere histone H3 gene ZmCENH3 in maize, and the OsCSN5 and OsPDCD5 genes in rice, are studied. The method of this invention allows for fine-tuning of their expression, providing a new technical approach for studying the function of these essential genes. Those skilled in the art can, based on the principles disclosed in this invention, design corresponding guide RNAs by performing sequence analysis on any target gene containing a conserved 5'-GT splicing site, to achieve targeted downregulation of the expression of that gene.

[0042] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A gene downregulation method based on regulating precursor mRNA cleavage efficiency, characterized in that, The method includes the following steps: S1. Identify the 5'-GT splicing site in the target gene: In the DNA sequence of the target gene, identify the 5' splicing site of at least one intron, wherein the 5' splicing site contains the GT dinucleotide sequence; S2. Design and Validation of Guide RNA: Design at least one guide RNA sequence that can target the 4th or 5th nucleotide downstream of the 5'-GT splicing site identified in S1, and verify that the guide RNA sequence has specific cleavage activity against the DNA fragment containing the site in a system containing Cas9 nuclease. S3. Constructing a gene editing vector: The guide RNA sequence verified in S2 is cloned into an expression vector containing the Cas9 nuclease coding sequence to construct a gene editing vector; S4. Genetic transformation and screening of mutants: The gene editing vector constructed in S3 is introduced into the target plant cells or tissues, and a transformed plant population is obtained through genetic transformation and regeneration. From the transformed plant population, mutant plants that have undergone site-directed editing at the 4th or 5th nucleotide downstream of the 5'-GT splice site are screened by DNA sequencing. S5. Transcript analysis: RNA was extracted from the mutant plants obtained in S4 and cDNA was obtained by reverse transcription; S6. Detecting transcript changes: Using the cDNA obtained in S5 as a template, PCR amplification is performed using primers that cross the editing site, and the amplification products are subjected to gel electrophoresis to obtain the transcript electrophoresis pattern of the target gene.

2. The gene downregulation method based on regulating precursor mRNA cleavage efficiency according to claim 1, characterized in that, In S1, the process of identifying the 5' splice site of at least one intron includes: Obtain the full-length DNA sequence of the target gene; In the full-length DNA sequence, the 5' boundaries of all introns were scanned and located one by one; Extract and record the nucleotide sequence starting at the 5' boundary of each intron, the sequence containing GT dinucleotide; From the recorded nucleotide sequences, 5' splicing sites containing conserved GUAAG or GTAA sequences were screened out.

3. The gene downregulation method based on regulating precursor mRNA cleavage efficiency according to claim 1, characterized in that, In S2, the process of designing at least one guide RNA sequence capable of targeting its downstream 4th or 5th nucleotide includes: Based on the nucleotide sequence of the selected 5' splice site, the positions of the 4th and 5th nucleotides downstream of it are determined; The target sequence is a sequence of at least 20 consecutive nucleotides containing the 4th or 5th nucleotide. The target sequence is linked to the guide RNA backbone sequence of the Cas9 nuclease to form a guide RNA sequence.

4. The gene downregulation method based on regulating precursor mRNA cleavage efficiency according to claim 1, characterized in that, In S2, the procedure for verifying that the guide RNA sequence has specific cleavage activity against DNA fragments containing that site in a system containing Cas9 nuclease includes: Construct a test expression vector containing the guide RNA sequence and the Cas9 nuclease encoding gene; The test expression vector was introduced into plant protoplasts for transient expression. Genomic DNA was extracted from the plant protoplasts and amplified by PCR using primers that span the 5'-GT splice site. If the PCR amplification product is detected and a smaller DNA fragment than the control is found, the guide RNA sequence is confirmed to have specific cleavage activity.

5. The gene downregulation method based on regulating precursor mRNA cleavage efficiency according to claim 1, characterized in that, In S3, the process of constructing a gene-editing vector includes: The constructed guide RNA sequence is inserted into a plant expression vector through an enzyme digestion and ligation reaction to form an intermediate vector containing a single guide RNA sequence expression cassette. The Cas9 nuclease coding sequence is inserted into a specific position in the intermediate vector through an enzyme digestion and ligation reaction, so that the guide RNA sequence expression cassette and the Cas9 nuclease coding sequence are in the same expression frame or a separate expression frame, thus constructing the gene editing vector.

6. The gene downregulation method based on regulating precursor mRNA cleavage efficiency according to claim 1, characterized in that, In step S4, the process of introducing the gene-editing vector constructed in step S3 into target plant cells or tissues, and obtaining a transformed plant population through genetic transformation and regeneration, includes: The constructed gene-editing vector was transformed into Agrobacterium competent cells to obtain engineered Agrobacterium; The engineered Agrobacterium was used to infect explants of the target plant; The infected explants were co-cultured and screened on a culture medium containing screening antibiotics to induce callus formation. The callus tissue was cultured on differentiation and rooting medium to regenerate complete transformed plants.

7. The gene downregulation method based on regulating precursor mRNA cleavage efficiency according to claim 1, characterized in that, In S4, the process of screening mutant plants from the transformed plant population by DNA sequencing for site-directed editing at the 4th or 5th nucleotide downstream of the 5'-GT splice site includes: Genomic DNA was extracted from the transformed plants; Using the genomic DNA as a template, PCR amplification was performed using the primer pairs shown in SEQ ID NO: 1 and SEQ ID NO: 2 to obtain a DNA fragment containing the 5'-GT splice site; The PCR amplification product was sequenced to obtain the nucleotide sequence of the 5'-GT splice site region; The obtained nucleotide sequence is compared with the unedited wild-type sequence. If the nucleotide at position 4 or 5 downstream of the 5'-GT splice site is deleted, inserted, or replaced, the plant is determined to be a site-edited mutant plant.

8. The gene downregulation method based on regulating precursor mRNA cleavage efficiency according to claim 1, characterized in that, In step S5, the process of extracting RNA from the mutant plant obtained in step S4 and reverse transcribing it to obtain cDNA includes: Tissues from the mutant plants identified as having undergone site-specific editing were collected, and total RNA was extracted using an RNA extraction kit. The concentration and purity of the extracted total RNA were determined; Using qualified total RNA as a template, reverse transcription was performed using reverse transcriptase and oligo(dT) or random primers to synthesize first-strand cDNA.

9. The gene downregulation method based on regulating precursor mRNA cleavage efficiency according to claim 1, characterized in that, In S6, the procedure for PCR amplification using the cDNA obtained in S5 as a template and primers spanning the edited site includes: Using the synthesized cDNA as a template, a primer pair consisting of the upstream primer shown in SEQ ID NO: 1 and the downstream primer shown in SEQ ID NO: 2 was used; PCR amplification was performed in a reaction system containing DNA polymerase, dNTPs, and magnesium ions.

10. The gene downregulation method based on regulating precursor mRNA cleavage efficiency according to claim 1, characterized in that, In step S6, the procedure for performing gel electrophoresis on the amplification products to obtain the transcript electrophoresis pattern of the target gene includes: Mix the obtained PCR amplification products with DNA loading buffer; The mixed sample was loaded into the wells of the agarose gel, and DNA molecular weight standards were loaded at the same time. Electrophoresis was performed at a constant voltage until the bromophenol blue indicator migrated to the vicinity of the bottom of the gel. The gel after electrophoresis was stained in nucleic acid staining solution and imaged under ultraviolet or blue light to obtain the transcript electrophoresis pattern showing DNA bands of different sizes.

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