Efficient CBE base editing tool based on Cas12i3 and application thereof

Abstract

The invention relates to the technical field of agriculture, in particular to an efficient CBE base editing tool based on Cas12i3 and application of the efficient CBE base editing tool. According to the Cas12i3-based efficient CBE base editing tool provided by the invention, the base editing tool comprises a protein expression cassette; the nucleotide sequence of the protein expression cassette is as shown in SEQ ID NO. 1. The tool can realize efficient, accurate and low-off-target C-to-T single base replacement in a plant genome, the editing efficiency and the target flexibility are remarkably improved, and an autonomous and controllable novel base editing platform is provided for crop functional gene research and molecular breeding.

Description

Technical Field

[0001] This invention relates to the field of agricultural technology, specifically to a highly efficient CBE base editing tool based on Cas12i3 and its applications. Background Technology

[0002] The key to crop genetic improvement lies in the effective creation and precise manipulation of genetic variation, a process that directly determines the efficiency and depth of new variety breeding. Traditional breeding methods mainly rely on variations generated by natural mutations or physicochemical mutagenesis, but these methods are characterized by significant randomness and uncertainty. In the vast crop genome, only a very small number of mutations can affect genes related to key traits, while most mutations may lead to loss of function or negative effects. Therefore, traditional breeding has inherent shortcomings in terms of efficiency, controllability, and goal orientation.

[0003] The emergence of gene editing technology has made it possible to achieve predictable and targeted precision modification at specific sites in the genome. The successful application of the CRISPR-Cas9 system in 2013 laid the foundation for molecular design breeding of crops. It introduces DNA double-strand breaks (DSBs) into target sites, and achieves gene knockout or targeted insertion through the cell's own repair mechanisms (mainly non-homologous end joining (NHEJ) or homologous recombination (HDR)). However, DSB-dependent editing still has the following limitations in plant cells: (1) the repair process is random, which can easily lead to irregular insertion or deletion mutations; (2) the editing efficiency is significantly affected by the cell repair pathway, especially the HDR pathway, which is extremely inefficient in plants; (3) repeated editing may cause chromosomal rearrangement and genome instability. To achieve higher precision genetic manipulation, base editing technology has been developed. This technology does not require DNA double-strand breaks, but achieves precise replacement of a single base by fusing a nicked nuclease or cleavage nuclease with a deaminase.

[0004] Compared to traditional gene knockout methods, base editing technology has advantages such as not relying on DSB (Digital Substance Sequence), controllable mutagenesis types, and stable and heritable mutations, making it a core tool for crop functional gene analysis and precision breeding. The CBE (Chemical Genetic Editing) system is particularly suitable for inducing conserved mutations, improving resistance genes, and simulating natural variation.

[0005] Currently, most mainstream CBE systems are built on the SpCas9 or LbCas12a framework, with APOBEC1, PmCDA1, or their modified evoCDA1 as the catalytic core. Although these systems have achieved C-to-T substitution in various plants, they still have the following shortcomings: 1. Unstable editing efficiency—APOBEC enzyme activity is significantly affected by DNA sequence context, with efficiency decreasing significantly in GC-rich regions or dense chromatin regions; 2. Prominent off-target and multi-site editing problems—tandem deamination is prone to occur in continuous cytosine sequences, leading to unintended mutations; 3. Limited PAM recognition—Cas9 only recognizes NGG, and Cas12a mainly recognizes TTTV, limiting the target coverage; 4. Obvious patent barriers—existing CBE systems are mostly subject to foreign core patents, hindering the establishment of independent technology systems. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a highly efficient CBE base editing tool based on Cas12i3 and its applications. This tool enables efficient, precise, and low-off-target C-to-T single-base substitutions in plant genomes, significantly improving editing efficiency and target flexibility, and providing a novel, autonomous, and controllable base editing platform for crop functional gene research and molecular breeding.

[0007] Therefore, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides, in optional embodiments, a highly efficient CBE base editing tool based on Cas12i3, the base editing tool comprising a protein expression cassette;

[0009] The nucleotide sequence of the protein expression cassette is shown in SEQ ID NO.1.

[0010] Preferably, the amino acid sequence of the protein expression cassette is shown in SEQ ID NO.2.

[0011] Preferably, the protein expression cassette includes a promoter, a 2×BP NLS nuclear localization signal fragment, cytosine deaminase Sdd7, a linker peptide, nCas12i3 (E844A) protein, and a terminator.

[0012] In this invention, the intermediate linker peptide is (Gly4Ser)6 or its functional homology sequence to ensure spatial independence of the catalytic and recognition domains. The promoter can be a 35S, Ubiquitin, or Actin promoter.

[0013] In this invention, Cas12i3 belongs to the CRISPR-Cas V-I system, possessing crRNA self-processing and highly specific DNA binding capabilities. The inactivated nCas12i3 (E844A) loses its endonuclease function but can still precisely locate the target DNA site. By fusing with a highly efficient Sdd7 deaminase, it catalyzes the deamination of cytosine (C) to uracil (U) in the single-stranded DNA region exposed at the target sequence. Uuracil is then replaced by thymine (T) during DNA replication or repair, achieving a single-base substitution C•G→T•A. This process does not require double-strand breaks, thus effectively avoiding insertion or deletion mutations and improving the stability and safety of editing. This invention introduces nuclear localization signals (NLS) at both ends of the fusion structure to ensure efficient aggregation of Sdd7-nCas12i3 in the cell nucleus and the execution of the deamination reaction. The length and sequence of the linker peptide are optimized to ensure that the Sdd7 active site can fully contact the target DNA region, thereby expanding the effective editing window and improving catalytic efficiency.

[0014] Preferably, in the protein expression box, from the N-terminus to the C-terminus, there are a promoter, a BP NLS nuclear localization signal fragment, a cytosine deaminase Sdd7, a linker peptide, an nCas12i3 protein, a BP NLS nuclear localization signal fragment, and a terminator, and a GCCACC kozak sequence is added to the 5' end of the cytosine deaminase Sdd7.

[0015] The Cas12i3 protein is a Cas12i3-E844A variant.

[0016] Preferably, the base editing tool further includes an RNA expression cassette;

[0017] The RNA expression cassette includes a promoter, a crRNA sequence, and a terminator;

[0018] The crRNA sequence consists of a DR sequence and a target-specific spacer sequence.

[0019] Preferably, the nucleotide sequence of the DR sequence is as shown in SEQ ID NO.3; and / or,

[0020] The target-specific spacer sequence is 20 nt in length and matches the target gene, which is selected from one of the rice BADH2 gene, rice OsPDS gene, rice OsALS gene, or rice OsSLR1 gene; and / or,

[0021] The DR sequence is located at the 5' end of the target-specific spacer sequence.

[0022] Preferably, when the target gene is the rice BADH2 gene, the nucleotide sequence of the target-specific spacer sequence is as shown in SEQ ID NO.4;

[0023] When the target gene is the rice OsPDS gene, the nucleotide sequence of the target-specific spacer sequence is shown in SEQ ID NO.5;

[0024] When the target gene is the rice OsALS gene, the nucleotide sequence of the target-specific spacer sequence is as shown in SEQ ID NO. 6;

[0025] When the target gene is the rice OsSLR1 gene, the nucleotide sequence of the target-specific spacer sequence is shown in SEQ ID NO.7.

[0026] Secondly, in an optional embodiment, the present invention provides an expression vector including the aforementioned efficient CBE base editing tool based on Cas12i3.

[0027] Thirdly, in an optional embodiment, the present invention provides a method for replacing base C with base T in a rice genome sequence, comprising the following steps:

[0028] The aforementioned efficient CBE base editing tool based on Cas12i3 was introduced into rice or rice cells to perform base editing on target genes in the rice genome, replacing the C base with the T base in the target genes of the rice genome.

[0029] Furthermore, in an optional embodiment, the present invention also provides a method for introducing a targeting vector into rice cells using the above-described expression vector, comprising the following steps:

[0030] (1) After removing the husks and sterilizing the Nipponbare rice seeds, the embryos were separated and placed on callus induction medium to generate secondary callus.

[0031] (2) Transfer the secondary callus to a new callus induction medium for pre-culture;

[0032] (3) Contact the callus obtained in step (2) with the Agrobacterium of the above expression vector corresponding to the target gene for 15 minutes.

[0033] (4) Transfer the callus tissue from step (3) to a culture dish with three sterile filter papers on it, and add 2.5-3.5 mL of Agrobacterium suspension culture medium to the culture dish and incubate at 21-23℃ for 48 hours;

[0034] (5) Place the callus tissue from step (4) on a pre-screening medium and culture for 5-7 days;

[0035] (6) Transfer the callus tissue from step (5) onto a screening medium to obtain resistant callus tissue;

[0036] (7) The resistant callus tissue was transferred to a differentiation and regeneration medium to differentiate into seedlings;

[0037] (8) Transfer the seedlings from step (7) to a rooting medium to root;

[0038] The target gene is selected from one of the following: rice BADH2 gene, rice OsPDS gene, rice OsALS gene, or rice OsSLR1 gene.

[0039] Fourthly, in an optional embodiment, the present invention provides an application of the above-mentioned efficient CBE base editing tool based on Cas12i3 in plant genome editing.

[0040] Preferably, the plant is a monocotyledonous plant, preferably rice, and more preferably japonica rice.

[0041] The nucleotide sequence of SEQ ID NO.1 is shown below:

[0042]

[0043] The amino acid sequence of SEQ ID NO.2 is shown below:

[0044]

[0045] The nucleotide sequence of SEQ ID NO.3 is shown below:

[0046] AGAGAATGTGTGCATAGTCACAC.

[0047] The nucleotide sequence of SEQ ID NO.4 is shown below:

[0048] CCTACACAGCAATCTTTCCT.

[0049] The nucleotide sequence of SEQ ID NO.5 is shown below:

[0050] CCAAGACCTCCACTAGAAAA.

[0051] The nucleotide sequence of SEQ ID NO.6 is shown below:

[0052] CAGGAGACGCCCATAGTCGA.

[0053] The nucleotide sequence of SEQ ID NO.7 is shown below:

[0054] GCCGGCTGCCACCGCGTCCA.

[0055] Compared with the prior art, the present invention has one of the following beneficial effects:

[0056] 1. The base editing tool provided by this invention can achieve efficient, precise and low off-target C-to-T single base substitution in plant genomes, significantly improving editing efficiency and target flexibility, and providing a novel base editing platform that is autonomous and controllable for crop functional gene research and molecular breeding.

[0057] 2. This invention constructs an Sdd7-nCas12i3 base editing protein by fusing the highly active cytosine deaminase Sdd7 with the nicking enzyme nCas12i3 (E844A). This fusion structure retains the DNA-specific recognition ability of Cas12i3 while significantly improving C-to-T substitution efficiency and editing purity. The fusion gene, optimized with plant codons, was constructed into a plant expression vector, and its editing performance was verified in rice. Experimental results show that this system exhibits efficient and stable editing effects at multiple target sites, including BADH2, PDS, ALS, and SLR1, demonstrating good reproducibility and universality. Attached Figure Description

[0058] Figure 1 This is a schematic diagram of the plant expression vector Sdd7-nCas12i3 prepared in Example 1 of the present invention;

[0059] Figure 2 This demonstrates the efficiency of Sdd7-nCas12i3 in targeted genome editing within callus tissue in Example 3 of this invention.

[0060] Figure 3 The editing efficiency of Sdd7-nCas12i3 and APOEE-nCas12i3 in T0 plants in Example 4 of this invention is shown. Clean C-to-T indicates that only the target C-to-T base substitution is present, with no other mutations; Chimeric indicates chimeric mutation; byproduct indicates editing byproduct. Detailed Implementation

[0061] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0062] Those skilled in the art can make certain equivalent modifications and obvious improvements to this invention.

[0063] Example 1

[0064] Obtaining Sdd7-nCas12i3

[0065] Based on the codon bias of rice, the inventors optimized the gene sequences of the 2×BP NLS nuclear localization signal fragment, cytosine deaminase Sdd7, linker peptide, and nCas12i3 (E844A), and directly synthesized the Sdd7-nCas12i3 sequence, the nucleotide sequence of which is shown in SEQ ID NO.1. To facilitate subsequent vector construction, PstI and SacI restriction sites were introduced at the 5' and 3' ends of the fusion gene, respectively, and ligated with the full-length gold T vector to obtain the T-Sdd7-nCas12i3 intermediate vector.

[0066] The T-Sdd7-nCas12i3 plasmid was double-digested with PstI and SacI enzymes (purchased from Thermo Fisher Scientific), and the Sdd7-nCas12i3 fragment was recovered by agarose gel electrophoresis. Simultaneously, the laboratory-preserved pHUC-SpR vector was linearized with PstI and SacI enzymes, and the vector backbone was recovered (the SpR gene was excised along with the PstI / SacI enzymes). The recovered T-Sdd7-nCas12i3 fragment was ligated to the pHUC vector backbone using T4 DNA ligase (purchased from NEB) at 16°C for 6 hours to obtain the plant intermediate expression vector pHUC-Sdd7-nCas12i3.

[0067] Furthermore, a crRNA expression cassette was introduced into the intermediate vector via the HindIII restriction site. This cassette consists of a 35S-CMVL complex promoter, a spectinomycin SpR resistance gene, and an HSP terminator. The SpR resistance gene was replaced with a crRNA sequence (composed of a DR sequence and a target-specific spacer sequence corresponding to the target gene), resulting in the plant targeting vector pHUC420-Sdd7-nCas12i3, named Sdd7-nCas12i3.

[0068] Example 2

[0069] Construction of Sdd7-nCas12i3 base editor plant targeting vector

[0070] The rice genes OsBADH2, OsPDS, OsALS, and OsSLR1 were selected as target genes, and crRNA primers targeting specific sites of these four genes were designed. The primer sequences are shown in Table 1, where lowercase letters represent complementary residues of the BsaI enzyme site, and underlined portions represent DR sequences.

[0071]

[0072] The primers were annealed to form double-stranded crRNA fragments, which were then ligated into the Sdd7-nCas12i3 vector, linearized by BsaI digestion, to obtain targeting vectors Sdd7-nCas12i3-BADH2-T, Sdd7-nCas12i3-PDS-T, Sdd7-nCas12i3-ALS-T, and Sdd7-nCas12i3-SLR1-T, respectively, targeting the SpR resistance genes of the four genes mentioned above with base editing replacements. These vectors were then transformed into Agrobacterium tumefaciens strain EHA105 (preserved in the Rice Precision Breeding Laboratory of Anhui Agricultural University) using a liquid nitrogen freeze-thaw method for subsequent rice genetic transformation.

[0073] Example 3

[0074] Rice genetic transformation vector

[0075] 1. Induction and pre-culture of mature embryo callus

[0076] Select mature seeds of the Japonica rice variety Nipponbare. After removing the outer husk, select seeds that are plump, free from mold, and undamaged. Soak the seeds in 70% alcohol and gently shake for 90 seconds, then discard the alcohol. Add a 50% sodium hypochlorite solution containing 1 drop of Tween20 (the stock solution has an effective chlorine concentration ≥4%), and sterilize by shaking at 180 rpm for 45 minutes. Discard the sterilization solution, rinse the seeds 5-10 times with sterile water until no sodium hypochlorite odor remains, and finally soak in sterile water at 30°C overnight.

[0077] The following day, the embryo was separated along the aleurone layer of the seed using a sterile scalpel. The embryos were placed, scutellum-side up, on callus induction medium (composition shown in Table 1), with 12 embryos per dish. Callus was induced by dark incubation at 30°C. After approximately 14 days of incubation, spherical, coarse-textured, light yellow secondary callus tissues were selected and transferred to fresh callus induction medium. These were then pre-cultured in the dark at 30°C for 5 days. After the pre-culture, small, well-formed, and actively dividing callus granules were collected and placed in 50 mL sterile centrifuge tubes for later use.

[0078] 2. Infection and co-culture

[0079] Add the prepared Agrobacterium suspension to the centrifuge tube containing the prepared callus tissue, ensuring the callus tissue is completely submerged. Soak for 15 minutes, gently shaking every 5 minutes to ensure full contact between the callus tissue and the Agrobacterium. After soaking, discard the suspension, transfer the callus tissue to sterile filter paper, blot off any excess bacterial solution, and then dry the surface of the callus tissue with sterile air in a laminar flow hood. Place three sheets of sterile filter paper in a 100×25mm sterile culture dish, add 2.5mL of Agrobacterium suspension culture medium, and evenly disperse the dried callus tissue on the filter paper. Incubate at 23°C in the dark for 48 hours.

[0080] 3. Screening and Cultivation

[0081] After co-culture, the callus tissue was transferred to pre-selection medium and cultured in the dark at 30°C for 5 days to inhibit Agrobacterium growth and preliminarily screen for transformed cells. After pre-selection culture, the callus tissue was transferred to selection medium, with 25 cells per culture dish, and cultured in the dark at 30°C for 2-3 weeks. After the resistant callus tissue showed obvious growth (characterized by firm texture and bright color), subsequent differentiation operations were carried out.

[0082] 4. Differentiation and regeneration

[0083] Select 2-3 healthy, fresh resistant callus particles from each independent transformant and transfer them to differentiation and regeneration medium (composition shown in Table 1). Place 5 independent transformants in each culture dish. Place the culture dishes in a 28℃ light incubator with a light cycle of 16 hours of light and 8 hours of darkness, and a light intensity of 3000-6000 lx, and culture until the callus differentiates into green shoots.

[0084] 5. Rooting and Transplanting

[0085] When the differentiated green shoots grow to about 2 cm, select one robust seedling from each independent transformant and transfer it to rooting medium (composition shown in Table 1). Culture at 28°C under light (light conditions are the same as for the differentiation stage). After two weeks of culture, select healthy seedlings with well-developed root systems and a height of 8-10 cm. Gently rinse the roots with sterile water to remove residual culture medium, then transplant them into flowerpots filled with sterilized nutrient soil and place them in a greenhouse for routine management.

[0086] Example 4

[0087] Base editing efficiency determination

[0088] First, the base editing efficiency of Sdd7-nCas12i3 was detected in resistant callus tissue. Two hundred newly formed resistant callus tissues from each transformation vector were selected, and DNA was extracted from the callus tissue using the CTAB method. The primers listed in Table 2 were used for the first round of PCR amplification. The PCR reaction system was prepared according to standard methods, and the reaction program was as follows: 95℃ pre-denaturation for 2 minutes; 94℃ denaturation for 30 seconds, 62℃ annealing for 30 seconds, and 72℃ extension for 20 seconds, for a total of 30 cycles; and a final extension at 72℃ for 5 minutes. After PCR, 3 μL of the product was subjected to gel electrophoresis to ensure the amplification of bands. The amplified product was then purified and recovered using a DNA product purification kit, mixed, and used for library construction. Next-generation sequencing (NGS) was used to detect the editing efficiency in the population.

[0089]

[0090] This implementation case tested the C-to-T editing activity of Sdd7-Cas12i3 at four endogenous genomic targets in rice (BADH2-T, OsPDS-T, OsALS-T, and OsSLR1-T). The results showed that the C-to-T editing rate exceeded 30% at the OsPDS-T target; the editing rate exceeded 20% at the OsALS-T target; and significant editing activity was also observed at the BADH2-T and OsSLR1-T targets (e.g., ...). Figure 2(As shown). This indicates that Sdd7-nCas12i3, as a highly efficient CBE tool, possesses excellent C-to-T single-base editing performance at multiple endogenous target sites in rice, and can meet the precise editing needs for improving different agronomic traits.

[0091] To detect whether the Sdd7-nCas12i3 editing gene produced editing at the plant level, genomic DNA was first extracted from the leaves of 48 transgenic rice plants after transplantation using the CTAB method. Using the extracted DNA as a template, PCR amplification was performed to detect the editing status at each target site. The same operation was performed simultaneously with the control vector transformation event. The Hi-TOM primers used for amplifying the genomic segments containing the target sites are shown in Table 3 (forward primer 5' bridge sequence added: ggagtgagtacggtgtgc; reverse primer bridge sequence added: gagtggatgctggatgg).

[0092]

[0093] The mixed-sample library was constructed and sequenced by the China National Rice Research Institute. Sequencing data were compared with a reference genome (Nipponbare) to split the samples, and the genotype and C-to-T base substitutions of each target-corresponding individual plant were accurately determined. Results are shown in [link to results]. Figure 3 The results showed that Sdd7-nCas12i3 exhibited significant C-to-T editing activity in T0 generation seedlings targeting four endogenous rice targets: BADH2-T, OsPDS-T, OsALS-T, and OsSLR1-T. The OsALS-T target showed the highest total C-to-T editing rate at 58.2%, with a clean C-to-T editing rate (containing only target base substitutions, without other mutations) of 81.3% (47.3% / 58.2%). The OsSLR1-T target showed a total C-to-T editing rate of 51.7%, with a clean C-to-T editing rate of 79.5% (41.1% / 51.7%). The OsPDS-T and BADH2-T targets showed total C-to-T editing rates of 42.3% and 38.5%, respectively, with clean C-to-T editing rates exceeding 75% in both cases. Meanwhile, the chimeric rates of all four targets were below 8%, with the OsALS-T target showing the lowest chimeric rate at only 7.8%.

[0094] In addition, using the same vector construction method, the Sdd7 cytosine deaminase was replaced with the commonly used APOEE deaminase to form the base editing vector APOEE-nCas12i3. The editing effects of Sdd7-nCas12i3 and APOEE-nCas12i3 were compared in T0 seedlings of the same four targeting vectors.

[0095] Among the four endogenous rice targets (BADH2-T, OsPDS-T, OsALS-T, and OsSLR1-T), Sdd7-nCas12i3 exhibited significantly better C-to-T base editing performance than APOEE-nCas12i3. In terms of overall editing rate, Sdd7-nCas12i3 achieved rates of 38.5% (BADH2-T), 42.3% (OsPDS-T), 58.2% (OsALS-T), and 51.7% (OsSLR1-T) at each target, all significantly higher than APOEE-nCas12i3 (approximately 10%, 20%, 18%, and 15% at each target). In terms of clean C-to-T editing rate (containing only the target C-to-T substitution, with no other accompanying mutations), Sdd7-nCas12i3 performed better, with a percentage of over 75% for each target, reaching as high as 81.3% and 79.5% for OsALS-T and OsSLR1-T targets, respectively. In contrast, the clean editing rate of APOEE-nCas12i3 was generally below 35%. Furthermore, the chimerism rate of Sdd7-nCas12i3 was below 8% for all targets (the lowest being 7.8% for OsALS-T), significantly lower than that of APOEE-nCas12i3 (which had a chimerism rate exceeding 15% for some targets, such as OsPDS-T).

[0096] The above results demonstrate that Sdd7-nCas12i3, as a highly efficient CBE tool, exhibits not only high editing efficiency (total editing rate of 38.5%-58.2%) in editing T0 generation seedlings at multiple endogenous target sites in rice, but also excellent editing purity (clean editing rate exceeding 75%) and low chimerism rate (<8%). Therefore, the Sdd7-nCas12i3 base editor constructed in this invention can efficiently and accurately achieve C-to-T base substitution in the rice genome, providing a reliable technical tool for molecular breeding work such as rice aroma improvement (BADH2-T), photosynthetic trait regulation (OsPDS-T), herbicide resistance enhancement (OsALS-T), and plant height directional regulation (OsSLR1-T).

[0097] Although the principles of the present invention have been described in detail above with reference to preferred embodiments, those skilled in the art should understand that the above embodiments are merely illustrative explanations of the implementation of the present invention and are not intended to limit the scope of the present invention. The details in the embodiments do not constitute a limitation on the scope of the present invention. Any obvious changes, such as equivalent transformations or simple substitutions, based on the technical solutions of the present invention without departing from the spirit and scope of the present invention fall within the protection scope of the present invention.

Claims

1. A Cas12i3-based efficient CBE base editing tool, characterized in that, The base editing tool includes a protein expression box; The nucleotide sequence of the protein expression cassette is shown in SEQ ID NO.

1.

2. The Casl2i3-based high-efficiency CBE base editing tool of claim 1, wherein, The amino acid sequence of the protein expression cassette is shown in SEQ ID NO.

2.

3. The efficient CBE base editing tool based on Cas12i3 according to claim 1, characterized in that, The protein expression cassette includes a promoter, a 2×BP NLS nuclear localization signal fragment, cytosine deaminase Sdd7, a linker peptide, nCas12i3 protein, and a terminator.

4. The efficient CBE base editing tool based on Cas12i3 according to claim 3, characterized in that, In the protein expression cassette, from the N-terminus to the C-terminus are the promoter, BP NLS nuclear localization signal fragment, cytosine deaminase Sdd7, linker peptide, nCas12i3 protein, BP NLS nuclear localization signal fragment, and terminator, and the 5' end of the cytosine deaminase Sdd7 has a GCCACC kozak sequence added.

5. The efficient CBE base editing tool based on Cas12i3 according to claim 1, characterized in that, The base editing tool also includes an RNA expression frame; The RNA expression cassette includes a promoter, a crRNA sequence, and a terminator; The crRNA sequence consists of a DR sequence and a target-specific spacer sequence.

6. The efficient CBE base editing tool based on Cas12i3 according to claim 5, characterized in that, The nucleotide sequence of the DR sequence is shown in SEQ ID NO.3; and / or, The target-specific spacer sequence is 20 nt in length and matches the target gene, which is selected from one of the rice BADH2 gene, rice OsPDS gene, rice OsALS gene, or rice OsSLR1 gene; and / or, The DR sequence is located at the 5' end of the target-specific spacer sequence.

7. An expression carrier, characterized in that, Includes the efficient CBE base editing tool based on Cas12i3 as described in any one of claims 1-6.

8. A method for replacing base C with base T in a rice genome sequence, characterized in that, Includes the following steps: The efficient CBE base editing tool based on Cas12i3 as described in any one of claims 1-6 is introduced into rice or rice cells to perform base editing on target genes in the rice genome, thereby replacing the C base with the T base in the target genes of the rice genome.

9. The application of the efficient CBE base editing tool based on Cas12i3 as described in any one of claims 1-6 in plant genome editing.

10. The application according to claim 9, characterized in that, The plant is a monocotyledonous plant, preferably rice, and more preferably japonica rice.

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