Efficient ABE base editing tool based on Cas12i3 and application thereof

Abstract

The invention relates to the technical field of agriculture, in particular to an efficient ABE base editing tool based on Cas12i3 and application of the efficient ABE base editing tool. The Cas12i3-based efficient ABE base editing tool provided by the invention comprises a protein expression cassette and an RNA (Ribonucleic Acid) expression cassette, the protein expression cassette comprises a promoter, a 2 * BP NLS nuclear localization signal fragment, a 2 * adenine deaminase TadA8e, a connecting peptide, a Cas12i3 notch enzyme fragment and a terminator, the RNA expression cassette comprises a promoter, a crRNA sequence and a terminator; wherein the crRNA sequence is composed of a DR sequence and a target specific spacer sequence. The tool realizes efficient and specific single base substitution of A-to-G in a rice genome by utilizing the targeted binding characteristic of Cas12i3 and the efficient deamination activity of 2 * TadA8e, has the advantages of high editing efficiency, low off-target rate, wide application range and the like, provides an efficient and accurate technical means for plant functional genome research and molecular breeding, and has a wide application prospect. The creation of excellent rice varieties can be accelerated.

Description

Technical Field

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

[0002] The core of crop breeding lies in the efficient creation and precise utilization of genetic variation, a process that directly determines the depth and efficiency of variety improvement. In traditional breeding models, the source of genetic variation mainly relies on the long-term accumulation of natural mutations or artificial physical and chemical mutagenesis. However, such variations have significant randomness and uncertainty. In the vast genome, only a very small number of mutations can precisely act on the key genes controlling desirable agronomic traits, and most mutations may be accompanied by negative effects, resulting in an extremely scarce availability of effective variation resources. This severely restricts the acceleration of the breeding process and the precise improvement of target traits.

[0003] The advent of gene editing technology has brought a revolutionary change to crop breeding, especially the breakthrough application of the CRISPR-Cas9 system, which has enabled the targeted introduction of variations at specific sites in the genome, breaking the limitations of traditional breeding. However, the current mainstream application of gene editing systems in crop breeding is still mainly based on gene knockout through random insertion or deletion of small fragments. This type of editing method is more suitable for functional inactivation studies, but it is difficult to meet the needs of fine-tuning gene function.

[0004] While transgenic technology has shown potential in improving crop traits such as insect resistance and herbicide resistance, its application faces significant obstacles due to the need for stable integration of exogenous genes. This is compounded by stringent regulatory barriers and varying public perceptions of its safety. Traditional artificial breeding methods, which selectively retain superior mutants through phenotypic screening, are not only time-consuming (often requiring years or even decades) but also significantly susceptible to environmental interference, making precise manipulation of target genes difficult. While homologous recombination technology theoretically enables site-specific genome modification, its efficiency in plant cells is extremely low (typically below 0.1%), hindering its large-scale implementation in practice.

[0005] In-depth research has shown that the regulation of many key agronomic traits in crops (such as yield, quality, disease resistance, and stress tolerance) is often directly related to single-base variations within specific genes. Among these, adenine-to-guanine (A-to-G) base substitution is a widely existing and functionally significant type of variation in nature. However, existing base editing tools generally suffer from low editing efficiency, high off-target risk, and limited applicability in complex genomic regions when achieving precise A-to-G substitutions. Efficient and specific A-to-G editing tools remain relatively scarce.

[0006] The discovery of type VI CRISPR-Cas12i family proteins has provided a new direction for overcoming this challenge. Among them, Cas12i3, as a representative member of this family, possesses natural advantages as a high-quality gene editing tool: it can self-process crRNA without the need for tracrRNA, greatly simplifying the crRNA design and delivery process, and naturally supports multi-target editing characteristics, making multi-gene regulation of complex agronomic traits possible. More importantly, the Cas12i3 system can circumvent the core patent restrictions related to Cas9, and is expected to form an editing tool system with independent intellectual property rights, laying the foundation for independent innovation in crop breeding technology.

[0007] However, current adenine base editors (ABEs) based on Cas12i3 still face core bottlenecks, with generally low editing efficiency. In its natural state, the Cas12i3 protein lacks sufficient activity at key amino acid sites for nucleic acid interaction, resulting in base editing efficiency far lower than the mature Cas9 system. In dicotyledonous plants such as peppers and soybeans, effective editing events are even difficult to detect. The inherent defects in Cas12i3's nucleic acid binding and cleavage activity prevent it from efficiently opening the DNA double helix to expose single-stranded substrates, hindering the full release of the catalytic potential of 2×TadA8e. Ultimately, this results in ABE tools typically achieving an editing efficiency of less than 15% in crop cells, far from meeting the demands for efficient editing in breeding practices. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides a highly efficient ABE base editing tool based on Cas12i3 and its applications. This tool utilizes the targeted binding characteristics of the Cas12i3 nickase (Cas12i3-E844A) and the highly efficient deamination activity of 2×TadA8e to achieve efficient and specific single-base substitution of A-to-G in the rice genome. It boasts advantages such as high editing efficiency, low off-target rate, and wide applicability, providing an efficient and precise technical means for plant functional genomics research and molecular breeding, and accelerating the creation of superior rice varieties.

[0009] Therefore, the present invention provides the following technical solution:

[0010] In a first aspect, the present invention provides, in optional embodiments, a highly efficient ABE base editing tool based on Cas12i3, the base editing tool comprising a protein expression cassette and an RNA expression cassette;

[0011] The protein expression cassette includes a promoter, a 2×BP NLS nuclear localization signal fragment, a 2×adenine deaminase TadA8e, a linker peptide, a Cas12i3 nicking enzyme, and a terminator.

[0012] The RNA expression cassette includes a promoter, a crRNA sequence, and a terminator;

[0013] The crRNA sequence consists of a DR sequence and a target-specific spacer sequence.

[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 2×adenine deaminase TadA8e, a linker peptide, a Cas12i3 nickase, a BP NLS nuclear localization signal fragment, and a terminator, and a GCCACC kozak sequence is added to the 5' end of the 2×adenine deaminase TadA8e.

[0015] Preferably, the nucleotide sequence of the protein expression cassette is as shown in SEQ ID NO.1; and / or,

[0016] The amino acid sequence of the protein expression cassette is shown in SEQ ID NO.2.

[0017] Preferably, the nucleotide sequence of the DR sequence is as shown in SEQ ID NO.3; and / or,

[0018] 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 PDS gene, rice Pid3 gene, or rice ACC gene; and / or,

[0019] The DR sequence is located at the 5' end of the target-specific spacer sequence.

[0020] 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;

[0021] When the target gene is the rice PDS gene, the nucleotide sequence of the target-specific spacer sequence is as shown in SEQ ID NO.5;

[0022] When the target gene is the rice Pid3 gene, the nucleotide sequence of the target-specific spacer sequence is shown in SEQ ID NO. 6;

[0023] When the target gene is the rice ACC gene, the nucleotide sequence of the target-specific spacer sequence is shown in SEQ ID NO.7.

[0024] Secondly, in an optional embodiment, the present invention provides an expression cassette, characterized in that it includes the aforementioned efficient ABE base editing tool based on Cas12i3.

[0025] Thirdly, in an optional embodiment, the present invention provides an expression vector, characterized in that it includes the above-mentioned efficient ABE base editing tool based on Cas12i3.

[0026] Fourthly, in an optional embodiment, the present invention provides a method for replacing base A with base G in a rice genome sequence, comprising the following steps:

[0027] The aforementioned efficient ABE 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 base A with base G in the target genes of the rice genome.

[0028] Fifthly, in an optional embodiment, the present invention provides a method for introducing a targeting vector into rice cells using the above-described expression vector, comprising the following steps:

[0029] (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.

[0030] (2) Transfer the secondary callus to a new callus induction medium for pre-culture;

[0031] (3) Contact the callus obtained in step (2) with the Agrobacterium of the above expression vector corresponding to the target gene for 15 minutes.

[0032] (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;

[0033] (5) Place the callus tissue from step (4) on a pre-screening medium and culture for 5-7 days;

[0034] (6) Transfer the callus tissue from step (5) onto a screening medium to obtain resistant callus tissue;

[0035] (7) The resistant callus tissue was transferred to a differentiation and regeneration medium to differentiate into seedlings;

[0036] (8) Transfer the seedlings from step (7) to a rooting medium to root;

[0037] The target gene is selected from one of the following: rice BADH2 gene, rice PDS gene, rice Pid3 gene, or rice ACC gene.

[0038]

[0039] The "optimized N6 macroelements" mentioned in Table 1 refers to the N6 macroelements [NO 3- ] / [NH 4+ = 40mM / 10mM.

[0040] In a sixth aspect, the present invention provides, in optional embodiments, the application of the above-mentioned efficient ABE base editing tool based on nCas12i3 in plant genome editing.

[0041] Preferably, the plant is a monocotyledonous plant, preferably rice, and more preferably japonica rice.

[0042] The nucleotide sequence of SEQ ID NO.1 is shown below:

[0043]

[0044] The amino acid sequence of SEQ ID NO.2 is shown below:

[0045]

[0046] The nucleotide sequence of SEQ ID NO.3 is shown below:

[0047] AGAGAATGTGTGCATAGTCACAC.

[0048] The nucleotide sequence of SEQ ID NO.4 is shown below:

[0049] AGCACTGAAGAAGAAGCCAT.

[0050] The nucleotide sequence of SEQ ID NO.5 is shown below:

[0051] AGGTTTGATAGAAAACTGAA.

[0052] The nucleotide sequence of SEQ ID NO.6 is shown below:

[0053] GGTTCGATAGCAAAGATGCA.

[0054] The nucleotide sequence of SEQ ID NO.7 is shown below:

[0055] TGACTGGAAGAACTGTTGGA.

[0056] Compared with the prior art, the present invention has one of the following beneficial effects:

[0057] 1. The base editing tool provided by this invention utilizes the targeting binding characteristics of Cas12i3 and the efficient deamination activity of 2×TadA8e to achieve efficient and specific single base substitution of A-to-G in the rice genome. It has advantages such as high editing efficiency, low off-target rate and wide applicability, providing an efficient and precise technical means for plant functional genomics research and molecular breeding, and can accelerate the creation of superior rice varieties. Attached Figure Description

[0058] Figure 1 This is a schematic diagram of the structures of Cas12i3-2×TadA8e and Cas12i3-TadA8e synthesized in Example 1 of the present invention;

[0059] Figure 2 A schematic diagram of the plant expression vector Cas12i3-2×TadA8e prepared in Example 1 of this invention;

[0060] Figure 3 This refers to the targeted genome editing effects of Cas12i3-2×TadA8e and Cas12i3-TadA8e in callus tissue in Example 3 of the present invention. Figure 3a) NGS was used to determine the targeted genome editing efficiency of Cas12i3-2×TadA8e and Cas12i3-TadA8e in callus tissue; Figure 3 b represents the editing window for Cas12i3-2×TadA8e and Cas12i3-TadA8e targeting genome editing in callus tissue;

[0061] Figure 4 This describes the editing of Cas12i3-2×TadA8e and Cas12i3-TadA8e in T0 plants in Example 4 of the present invention. Figure 4 a represents the Hi-TOM assay used to determine the editing efficiency of Cas12i3-2×TadA8e and Cas12i3-TadA8e in T0 plants; Ho: homozygous mutation; Bi: biallelic mutation; HE: heterozygous mutation; Chi: chimeric mutation; WT: wild-type. Figure 4 b is a Sanger sequencing diagram showing the editing effects of Cas12i3-2×TadA8e and Cas12i3-TadA8e in T0 plants. Detailed Implementation

[0062] 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.

[0063] Those skilled in the art can make certain equivalent modifications and obvious improvements to this invention.

[0064] Unless otherwise specified, the operations described in the following embodiments are performed using conventional practices common in the art. Those skilled in the art can readily obtain instruction on such conventional practices from existing technologies, for example, by referring to textbooks such as Sambrook and David Russell, *Molecular Cloning: A Laboratory Manual*, 3rd ed., Vols 1, 2; and Charles Neal Stewart, Alisher Touraev, Vitaly Citovsky and Tzvi Tzfira, *Plant Transformation Technologies*. Unless otherwise specified, the medicinal materials, reagents, and other ingredients used in the following examples are commercially available products.

[0065] Example 1

[0066] Acquisition of Cas12i3-2×TadA8e

[0067] This invention first optimizes the sequences of elements such as double-copy TadA8e (2×TadA8e), Cas12i3 nickase (Cas12i3-E844A variant), 2×BP NLS nuclear localization signal fragment, and linker peptide using rice codons, assembling them into the Cas12i3-2×TadA8e sequence (see...). Figure 1 The sample was then sent to the company for synthesis. To facilitate subsequent vector construction, PstI and SacI restriction sites were introduced at the 5' and 3' ends of Cas12i3-2×TadA8e, respectively, and then inserted into the T vector to obtain the T-Cas12i3-2×TadA8e intermediate vector.

[0068] Meanwhile, using the same method described above, a T-Cas12i3-TadA8e intermediate vector containing only one TadA8e deaminase was synthesized (see [link to original text]). Figure 1 This is used to compare whether concatenating two TadA8e values ​​improves the editing efficiency of A-to-G.

[0069] From Escherichia coli XL-blue containing the T-Cas12i3-2×TadA8e vector, plasmids were extracted using the Tiangen plasmid extraction kit after kanamycin resistance screening and colony PCR identification. The plasmids were double-digested with PstI / SacI enzymes (purchased from Thermo Fisher Scientific), and the Cas12i3-2×TadA8e fragment was recovered by agarose gel electrophoresis. Simultaneously, the laboratory-preserved pHUC SpR vector was linearized using PstI / SacI enzyme, and the vector backbone was recovered (the SpR gene was excised along with the PstI / SacI enzyme). The recovered T-Cas12i3-2×TadA8e fragment was ligated to the pHUC vector backbone using T4 DNA ligase (purchased from NEB) at 16℃ for 6 h to obtain the plant intermediate expression vector pHUC-Cas12i3-2×TadA8e. Similarly, pHUC-Cas12i3-TadA8e was obtained. The Cas12i3-2×TadA8e and Cas12i3-TadA8e fragments were driven by the Ubi promoter, and the terminator used was the 35S terminator.

[0070] Furthermore, a crRNA expression cassette, consisting of a 35S-CMVL complex promoter, a spectinomycin SpR resistance gene, and an HSP terminator, was introduced into the intermediate vector via the HindIII restriction site. 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), thus constructing the plant targeting vector pHUC420-Cas12i3-2×TadA8e, named Cas12i3-2×TadA8e (see [link to documentation]). Figure 2Similarly, pHUC420-Cas12i3-TadA8e was obtained and named Cas12i3-TadA8e. All intermediate and final vectors involved in this implementation were subjected to Sanger sequencing to confirm sequence accuracy before proceeding to the next experimental step.

[0071] Example 2

[0072] Construction of the Cas12i3-2×TadA8e base editor plant targeting vector

[0073] The rice genes BADH2, PDS, Pid3, and ACC were selected as target genes, and crRNA sequences targeting specific sites of these four genes were designed. Primer sequences are shown in Table 2, where lowercase letters represent complementary residues at the BsaI enzyme site, and underlined portions represent DR sequences.

[0074] The primers were annealed to form double-stranded crRNA fragments, which were then ligated with the enzyme-digested and linearized Cas12i3-2×TadA8e and control vectors Cas12i3-TadA8e. After Sanger sequencing and confirmation of correct alignment, the targeting vectors Cas12i3-2×TadA8e-BADH2-T, Cas12i3-2×TadA8e-PDS-T, Cas12i3-2×TadA8e-Pid3-T, and Cas12i3-2×TadA8e-ACC-T targeting the above four genes were obtained; as well as four control vectors Cas12i3-TadA8e-BADH2-T, Cas12i3-TadA8e-ACC-T, Cas12i3-TadA8e-PDS-T, and Cas12i3-TadA8e-Pid3-T were obtained.

[0075] The vector was transformed into Agrobacterium tumefaciens strain EHA105 using the liquid nitrogen freeze-thaw method for subsequent genetic transformation in rice.

[0076]

[0077] Example 3

[0078] Detection of the efficiency of rice genetic transformation and resistant callus editing using targeting vectors.

[0079] 1. Induction and pre-culture of mature embryo callus

[0080] Mature seeds of the Japonica rice variety Nipponbare were selected. After removing the outer husk, seeds that were plump, free from mold, and undamaged were selected. The seeds were soaked in 70% alcohol and gently shaken for 90 seconds, then the alcohol was discarded. A 50% sodium hypochlorite solution containing 1 drop of Tween20 (with an effective chlorine concentration ≥4%) was then added, and the solution was placed on a shaker at 180 rpm for 45 minutes for sterilization. The sterilization solution was discarded, and the seeds were rinsed 5-10 times with sterile water until no sodium hypochlorite odor remained. Finally, sterile water was added, and the seeds were soaked overnight at 30°C.

[0081] 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.

[0082] 2. Cultivation of Agrobacterium strains and preparation of suspension

[0083] Agrobacterium strain EHA105 containing the targeting vector was streaked on LB solid medium containing 50 mg / L kanamycin (components shown in Table 1) and incubated in the dark at 28°C for 24 hours for the first activation. Then, a single colony was picked with a sterile inoculation loop and inoculated onto fresh LB solid medium containing 50 mg / L kanamycin and incubated in the dark at 28°C overnight for the second activation.

[0084] Take a 50mL sterile centrifuge tube, add 20-30mL of Agrobacterium suspension medium (components are shown in Table 1), scrape off the Agrobacterium colonies that have been activated for the second time with an inoculation loop, add them to the suspension medium, mix gently, measure the OD660 value with a spectrophotometer, adjust it to 0.10-0.25, let it stand at room temperature for more than 30 minutes, and set aside for later use.

[0085] 3. Infection and co-culture

[0086] 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 moisture of the callus tissue with sterile air in a laminar flow hood.

[0087] Three sheets of sterile filter paper were placed in a 100×25mm sterile culture dish, and 2.5mL of Agrobacterium suspension medium was added. The dried callus tissue was evenly dispersed on the filter paper and cultured for 48 hours in the dark at 23℃.

[0088] 4. Pre-screening and screening culture

[0089] After co-culture, the callus tissue was transferred to pre-selection medium (components shown in Table 1) 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 (components shown in Table 1), 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 performed.

[0090] Meanwhile, 200 newly formed resistant callus tissues were selected from each transformation vector to form a population; DNA was extracted from the callus tissue using the CTAB method; the first round of PCR amplification was performed using primers listed in Table 2. 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, 72℃ extension for 20 seconds, for a total of 30 cycles; and a final extension at 72℃ for 10 minutes. After PCR, 3 μL of the product was subjected to gel electrophoresis to ensure the amplification of bands; then, the amplified products were purified and recovered using a DNA product purification kit, and a library was constructed. Next-generation sequencing (NGS) was used to detect the editing efficiency in the population. NGS sequencing primers are shown in Table 3.

[0091]

[0092] This implementation case tested and compared all mutations of Cas12i3-2×TadA8e and the control vector Cas12i3-TadA8e within a 20bp target range on four endogenous genes in rice. The results showed that the A-to-G editing efficiency of Cas12i3-TadA8e at the four target sites (BADH2-T, Pid3-T, PDS-T, and ACC-T) ranged from 6.01% to 24.22%, with an average efficiency of 10.61% (see [link to implementation details]). Figure 3 In contrast, Cas12i3-2×TadA8e exhibited significantly higher activity, reaching a maximum efficiency of 35.93% at PDS-T sites, and the average efficiency of Cas12i3-2×TadA8e was also improved to 22.95%, which is 2.16 times higher than that of Cas12i3-TadA8e (Figure 3a), and the editing was mainly limited to a 4 bp window of positions 9-12. Figure 3 (b) With the improved precision base editing efficiency of Cas12i3-2×TadA8e, no unexpected byproducts were detected in the editing results. This indicates that Cas12i3-2×TadA8e, as an optimized editing tool, has superior editing performance on most endogenous targets in rice.

[0093] Example 4

[0094] Obtaining and identifying mutants of the targeting vector

[0095] 1. Differentiation and regeneration

[0096] 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.

[0097] 2. Rooting and Transplanting

[0098] 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.

[0099] 3. Molecular identification

[0100] Genomic DNA was extracted from the leaves of 48 transgenic rice plants after transplantation using the CTAB method. The extracted DNA was used as a template for PCR amplification to detect the editing status of each target site. The same operation was performed simultaneously with the control vector transformation event. The Hi-TOM primers used for amplifying the genomic regions containing the target sites are shown in Table 4 (forward primer with a bridge sequence added to the 5' end: ggagtgagtacggtgtgc; reverse primer with a bridge sequence added: gagtggatgctggatgg). The PCR reaction system was prepared according to Example 3.

[0101]

[0102] Since the four vectors target different genomic sequences, the amplification products were mixed and sequenced. The sequencing results were then aligned to a reference genome for analysis, and the genotype of each plant corresponding to each target was determined. A-to-G base substitutions were also analyzed. In T0 generation seedlings of the four rice endogenous targets (BADH2-T, Pid3-T, PDS-T, and ACC-T), the average A-to-G base substitutions of Cas12i3-2×TadA8e were 60.42%, 79.17%, 58.33%, and 60.42%, respectively, all significantly higher than those of Cas12i3-TadA8e (see [link to relevant documentation]). Figure 4 Furthermore, compared to the small number of homozygous A-to-G base substitution lines produced by Cas12i3-TadA8e (average 1.56% across all target sites), Cas12i3-2×TadA8e produced homozygous or biallelic edits at an average frequency of 10.42%, particularly at the PDS-T site, reaching a maximum of 20.83%. These results indicate that Cas12i3-based editors have high practicality and potential in plant adenine base editing.

[0103] 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 highly efficient ABE base editing tool based on Cas12i3, characterized in that, The base editing tools include a Cas protein expression box and an RNA expression box; The Cas protein expression cassette includes a promoter, a 2×BP NLS nuclear localization signal fragment, a 2×adenine deaminase TadA8e, a linker peptide, a Cas12i3 nicking enzyme, and a terminator. 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.

2. The efficient ABE base editing tool based on Cas12i3 according to claim 1, characterized in that, In the protein expression box, from the N-terminus to the C-terminus are the promoter, BP NLS nuclear localization signal fragment, 2×adenine deaminase TadA8e, linker peptide, Cas12i3 nickase, BP NLS nuclear localization signal sequence and terminator, and the 5' end of the 2×adenine deaminase TadA8e is supplemented with the kozak sequence of GCCACC. The Cas12i3 nickase is a Cas12i3-E844A variant; The linker peptide is a 6×GGGGS linker peptide.

3. The efficient ABE base editing tool based on Cas12i3 according to claim 1, characterized in that, The nucleotide sequence of the protein expression cassette is shown in SEQ ID NO.1; and / or, The amino acid sequence of the protein expression cassette is shown in SEQ ID NO.

2.

4. The efficient ABE base editing tool based on Cas12i3 according to claim 1, 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 PDS gene, rice Pid3 gene, or rice ACC gene; and / or, The DR sequence is located at the 5' end of the target-specific spacer sequence.

5. An expression box, characterized in that, Includes the efficient ABE base editing tool based on Cas12i3 as described in any one of claims 1-4.

6. An expression carrier, characterized in that, Includes the efficient ABE base editing tool based on Cas12i3 as described in any one of claims 1-4.

7. A method for replacing base A with base G in a rice genome sequence, characterized in that, Includes the following steps: The efficient ABE base editing tool based on Cas12i3 as described in any one of claims 1-4 is introduced into rice or rice cells to perform base editing on target genes in the rice genome, thereby replacing base A with base G in the target genes in the rice genome.

8. A method for introducing a targeting vector into rice cells using the expression vector according to claim 6, characterized in that, Includes the following steps: (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. (2) Transfer the secondary callus to a new callus induction medium for pre-culture; (3) Contact the callus obtained in step (2) with the Agrobacterium of the expression vector of claim 6 corresponding to the target gene for 15 minutes. (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; (5) Place the callus tissue from step (4) on a pre-screening medium and culture for 5-7 days; (6) Transfer the callus tissue from step (5) onto a screening medium to obtain resistant callus tissue; (7) The resistant callus tissue was transferred to a differentiation and regeneration medium to differentiate into seedlings; (8) Transfer the seedlings from step (7) to a rooting medium to root; The target gene is selected from one of the following: rice BADH2 gene, rice PDS gene, rice Pid3 gene, or rice ACC gene.

9. The application of the efficient ABE base editing tool based on Cas12i3 as described in any one of claims 1-4 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.

Images