Editor fusing 5 'exonuclease and CRISPR-Cas12i2 as well as construction and application of editor

Abstract

The invention discloses an editor fusing 5 'exonuclease and CRISPR-Cas12i2 as well as construction and application of the editor, and relates to the technical field of plant biology. The editor for fusing the 5'exonuclease and the CRISPR-Cas12i2 comprises a 5 'exonuclease T5E and Cas12i2 variant fusion protein T5E-Cas12i2 v1 coded by a nucleotide sequence, and a nuclear localization signal bpNLS, the 5' exonuclease T5E, the Cas12i2 variant Cas12i2 v1 and a nuclear localization signal bpNLS are sequentially arranged from the N end to the C end. Compared with an existing plant genome editing tool, the constructed T5E-Cas12i2v1 editor has the advantages that the efficiency is high, TTN-PAM sites are targeted, and site-specific large fragment deletion can be generated. The method is beneficial to accurate manipulation research on large cis-genetic elements, and has a wide application prospect in plant functional genome research and genetic improvement of crops.

Description

Technical Field

[0001] This invention relates to the field of plant biotechnology, and more particularly to an editor that integrates a 5′ exonuclease and CRISPR-Cas12i2, its construction, and its application. Background Technology

[0002] Against the backdrop of multiple challenges, including global population growth, escalating climate change, and arable land scarcity, agricultural production faces unprecedented pressure. Rapidly developing high-yield, high-quality, and stress-resistant crop varieties through advanced biotechnology has become a core requirement for ensuring food security and ecological balance. In recent years, CRISPR-Cas gene editing technology, with its precise and efficient gene modification capabilities, has become an indispensable core tool in plant functional genomics research and crop genetic improvement. Among them, the Cas12 effector subfamily, belonging to class II type V, has attracted much attention due to its series of unique biological characteristics. This subfamily includes several members such as Cas12a, Cas12c1, Cas12i2, Cas12i3, and Cas12j. Compared to other Cas effectors, members of the Cas12 family exhibit three core advantages: 1. They can generate staggered double-strand breaks when cutting target DNA; 2. They have a relatively compact molecular structure, which makes them suitable for delivery in traditional transformation systems such as plant protoplast transformation and Agrobacterium-mediated transformation, lowering the technical threshold for delivery of editing tools; 3. They possess unique requirements for protospacer adjacent motif (PAM) recognition, and the PAM specificity varies among different members, greatly expanding the range of target sites that can be selected.

[0003] Cas12i2, a novel Cas12 effector discovered in recent years, further enriches the CRISPR-Cas editing toolkit with its unique molecular functions. Existing research has confirmed that Cas12i2 possesses the ability to recognize 5′-TTN-rich PAM sequences. This PAM specificity allows it to complement other Cas12 members in target selection, enabling it to target more gene sites that are difficult for traditional tools to access. Simultaneously, in vitro biochemical experiments and in vivo experiments in mammalian cell lines have demonstrated that Cas12i2 exhibits highly efficient DNA editing activity, enabling precise cutting and modification of target genes, showcasing its potential as an editing tool. However, although Cas12i2 has been preliminarily validated in in vitro systems and mammalian models, its application potential in plant genome engineering remains largely unexplored. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide an editor that integrates a 5′ exonuclease and CRISPR-Cas12i2, as well as its construction and application, to solve the problems of low editing efficiency and unclear editing characteristics of plant Cas12i2 in the prior art. Through engineering, a Cas12i2 v1 variant with high editing activity is obtained, and its editing efficiency is further improved and large fragment deletions are promoted by integrating the 5′ exonuclease T5E.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: The first objective of this invention is to provide a fusion protein T5E-Cas12i2 v1, a 5′ exonuclease and CRISPR-Cas12i2 variant for plant gene editing.

[0006] A second objective of the present invention is to provide a multinucleotide sequence encoding the 5′ exonuclease and CRISPR-Cas12i2 variant fusion protein T5E-Cas12i2 v1.

[0007] A third object of the present invention is to provide a plasmid vector containing the said polynucleotide sequence.

[0008] A fourth object of the present invention is to provide the use of the 5′ exonuclease and CRISPR-Cas12i2 variant fusion protein T5E-Cas12i2 v1 or the polynucleotide sequence or the plasmid vector in the preparation of a plant T5E-Cas12i2 v1 editor.

[0009] The fifth objective of this invention is to provide a plant T5E-Cas12i2 v1 editor.

[0010] The sixth object of the present invention is to provide a method for constructing the plant T5E-Cas12i2 v1 editor.

[0011] A seventh object of the present invention is to provide the application of the plant T5E-Cas12i2 v1 editor in plant gene editing.

[0012] The above-mentioned objective of this invention is achieved through the following technical solution: A fusion protein T5E-Cas12i2 v1, a 5′ exonuclease and CRISPR-Cas12i2 variant for plant gene editing, comprises, from the N-terminus to the C-terminus, the 5′ exonuclease T5E and the Cas12i2 enzyme variant Cas12i2 v1. The amino acid sequence of the 5′ exonuclease T5E is shown in SEQ ID NO. 6, and the amino acid sequence of the Cas12i2 enzyme variant Cas12i2 v1 is shown in SEQ ID NO. 7.

[0013] Preferably, the 5′ exonuclease-CRISPR-Cas12i2 variant fusion protein T5E-Cas12i2 v1 further includes two nuclear localization signals fused to the N-terminus and C-terminus of T5E-Cas12i2 v1, respectively. The function of these nuclear localization signals is to allow the fusion protein to be correctly located in the cell nucleus for nuclear genome editing.

[0014] More preferably, the nuclear localization signal is a highly efficient nuclear localization signal bpNLS, whose amino acid sequences at the N-terminus and C-terminus of the fusion protein are shown in SEQ ID NO.8 and SEQ ID NO.9, respectively.

[0015] Preferably, the 5′ exonuclease and CRISPR-Cas12i2 variant fusion protein T5E-Cas12i2 v1 further includes fusion protein adapter sequences that link the 5′ exonuclease T5E and the Cas12i2 v1 variant, respectively. Its function is to allow the T5E and Cas12i2 v1 dual protein monomers to spatially combine into a form suitable for gene editing. Theoretically, the protein adapter sequence includes any sequence that does not interfere with the function of the target protein.

[0016] More preferably, the linker sequence is a flexible linker sequence, specifically linker1, whose amino acid sequence is shown in SEQ ID NO.10.

[0017] Preferably, the 5′ exonuclease and the CRISPR-Cas12i2 variant fusion protein T5E-Cas12i2 v1, from the N-terminus to the C-terminus, sequentially includes the nuclear localization signal bpNLS1, the 5′ exonuclease T5E, the flexible linker sequence linker1, the Cas12i2 enzyme variant Cas12i2 v1, and the nuclear localization signal bpNLS2, i.e., fused in the order of bpNLS1-T5E-linker1-Cas12i2 v1-bpNLS2, and its full amino acid sequence is shown in SEQ ID NO.12.

[0018] In this context, bpNLS1 and bpNLS2 are both nuclear positioning signals bpNLS, with bpNLS1 representing the first bpNLS and bpNLS2 representing the second bpNLS.

[0019] A polynucleotide sequence encoding the fusion protein T5E-Cas12i2 v1 of the 5′ exonuclease and CRISPR-Cas12i2 variant described in any of the above.

[0020] Preferably, the nucleotide sequence encoding the 5′ exonuclease T5E is shown in SEQ ID NO.1, and the nucleotide sequence encoding the Cas12i2 enzyme variant Cas12i2 v1 is shown in SEQ ID NO.2, both optimized according to rice codon preferences.

[0021] Preferably, the nucleotide sequences encoding the nuclear localization signals bpNLS at the N-terminus and C-terminus of the fusion protein are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively, and are optimized according to the codon preference of rice.

[0022] Preferably, the nucleotide sequence encoding the flexible linker sequence connecting the 5′ exonuclease T5E and the Cas12i2 enzyme variant Cas12i2 v1, as shown in SEQ ID NO.5, is optimized according to rice codon preferences.

[0023] More preferably, the polynucleotide sequence, as shown in SEQ ID NO.11, is fused in the order bpNLS-T5E-linker1-Cas12i2 v1-bpNLS, and all components are optimized according to rice codon preferences.

[0024] The present invention also provides plasmid vectors containing any of the polynucleotide sequences described above.

[0025] The present invention also provides the use of the 5′ exonuclease and CRISPR-Cas12i2 variant fusion protein T5E-Cas12i2 v1 or any of the polynucleotide sequences or plasmid vectors described above in the preparation of a plant T5E-Cas12i2 v1 editor.

[0026] A plant T5E-Cas12i2 v1 editor is obtained by constructing a polynucleotide sequence encoding the fusion protein T5E-Cas12i2 v1 of any of the above-mentioned 5′ exonuclease and CRISPR-Cas12i2 variant into a plant transformation vector.

[0027] Preferably, the plant transformation vector is a binary vector pYLCRISPR-Cas9Pubi-H.

[0028] The present invention also provides a method for constructing the plant T5E-Cas12i2 v1 editor, which first prepares a complete fusion DNA sequence encoding the fusion protein T5E-Cas12i2 v1 of a 5′ exonuclease and a CRISPR-Cas12i2 variant, and then assembles it between Pst I and BamH I of the binary vector pYLCRISPR-Cas9Pubi-H to obtain the editor T5E-Cas12i2 v1.

[0029] The present invention also provides the application of the plant T5E-Cas12i2 v1 editor in plant genome editing.

[0030] Preferably, the plant is rice.

[0031] The beneficial effects of this invention include at least the following: This invention provides a plant CRISPR-Cas12i2 editor, T5E-Cas12i2 v1, which is highly efficient, targets the TTN-PAM site, and can produce site-specific large deletions. It also provides a plant T5E-Cas12i2 v1 editing system based on this fusion protein. This overcomes the limitations of typical CRISPR-Cas9, which cannot target AT-rich genomic regions, and the low editing efficiency and short deletion lengths of ordinary CRISPR-Cas12i2. This system is more beneficial for research on plant gene function and for crop genetic improvement. Attached Figure Description

[0032] Figure 1 Analysis of the structure and editing characteristics of the plant Cas12i2 v1 and T5E-Cas12i2 v1 editor vectors. (A) Map of the Cas12i2 v1 and T5E-Cas12i2 v1 editor vectors. (B) Statistical graph of the editing efficiency of the Cas12i2 v1 and T5E-Cas12i2 v1 editors at 12 target sites, and a presentation of their editing efficiency at four different types of PAM sites. (C) Detailed target site sequences and editing efficiency statistics of the Cas12i2 v1 and T5E-Cas12i2 v1 editors at 12 target sites, with PAMs underlined.

[0033] Figure 2 This refers to the tandem crRNA expression cassette sequence for the Cas12i2 v1 and T5E-Cas12i2 v1 editors. To facilitate the in vivo processing of the tandem crRNA expression cassette into a single crRNA for functional purposes, this invention uses a tRNA-crRNA-HDV tandem structure. Italics indicate the tRNA sequence, bold text indicates crRNA, underline indicates the target sequence, and gray text indicates the HDV sequence.

[0034] Figure 3 A comparison of large fragment missing patterns generated by the Cas12i2 v1 and T5E-Cas12i2 v1 editors. (A) Statistics on the probability of different length missing ranges generated by the Cas12i2 v1 and T5E-Cas12i2 v1 editors. (B) Detailed statistical table of different length missing ranges generated by the Cas12i2 v1 and T5E-Cas12i2 v1 editors.

[0035] Figure 4 Analysis of mutation types generated by the Cas12i2 v1 and T5E-Cas12i2 v1 editors at two representative target sites, TS4 and TS11. Underlined sequences represent PAM sequences, bolded sequences represent target sequences, and deleted fragments are indicated by horizontal lines. (A) TS4 site. (B) TS11 site.

[0036] Figure 5 To investigate the directional manipulation and function of the rice endogenous transposon element MITE using the T5E-Cas12i2 v1 editor. (A) Schematic diagram of directional deletion of the MITE transposon element on the promoter of the rice endogenous gene OsDOG using the T5E-Cas12i2 v1 editor. (B) Identification of plants with homozygous deletion of MITE using specific primers and PCR. (C) Sanger sequencing identification of PCR for homozygous deletion of MITE.

[0037] Figure 6 Morphological observation of seedlings of MITE-edited plant DOGmite and wild-type ZH11. (A) Seedling height phenotype of wild-type ZH11, non-MITE homozygous deletion line L3 (negative control), and MITE homozygous deletion lines L7 and L17. Scale bar is 1 cm. (B) Plant height statistics of ZH11 and MITE-edited lines. (C) Endogenous OsDOG gene expression analysis of ZH11 and MITE-edited lines. Significant differences were indicated between different letters (Duncan's method analysis, P < 0.05). Detailed Implementation

[0038] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0039] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0040] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0041] The following specific embodiments illustrate the solution proposed in this invention: Example 1: Construction of Plant Cas12i2 v1 and T5E-Cas12i2 v1 Editor The protein sequences of the 5′ exonuclease T5E, the Cas12i2 variant Cas12i2 v1, and bpNLS were directly synthesized based on published sequences (Ceska et al., 1996, Nature, 382:90–93; McGaw et al., 2022, Nature Communications, 13:2833; Thuronyie et al., 2019, Nature Biotechnology, 37:1070–1079), using two fragments: bpNLS-T5E-linker1 and Cas12i2 v1-bpNLS, optimized according to rice codon preferences. The optimized nucleic acid sequences of T5E, Cas12i2 v1, N-terminal bpNLS, C-terminal bpNLS, and linker1 are shown in SEQ ID NO. 1–5, and their encoded amino acid sequences are shown in SEQ ID NO. 6–10. SEQ ID NO.1 (DNA sequence of the 5′ exonuclease T5E functional component, 870 bp): TCCAAGAGCTGGGGCAAGTTCATCGAGGAGGAGGAGGCCGAGATGGCGAGCAGGAGGAACCTCATGATCGTCGATGGCACCAACCTGGGCTTCCGCTTCAAGCATAACAATAGCAAGAAGCCGTTCGCCAGCAGCTACGTGTCCACCATCCAGAGCCTCGCCAAGAGCTACTCCGCCCGCACCACCATCGTCCTCGGCGATAAGGGCAAGAGCGTCTTCAGGCTGGAGCACCTGCCGGAGTACAAGGGCAATCGCGACGAGAAGTACGCGCAGAGGACCGAGGAGGAGAAGGCCCTCGACGAGCAGTTCTTCGAGTACCTGAAGGACGCCTTCGAGCTGTGCAAGACCACCTTCCCGACCTTCACCATCCGCGGCGTGGAGGCCGACGACATGGCCGCTTACATCGTCAAGCTCATCGGCCATCTGTACGACCACGTCTGGCTCATCTCCACCGATGGCGATTGGGACACCCTCCTCACCGATAAGGTGAGCAGGTTCTCCTTCACCACCCGCAGGGAGTACCACCTGCGCGACATGTACGAGCATCACAATGTCGACGATGTGGAGCAGTTCATCAGCCTCAAGGCGATCATGGGCGACCTGGGCGATAACATCAGGGGCGTGGAGGGCATCGGCGCCAAGAGGGGATACAATATCATCAGGGAGTTCGGCAATGTGCTGGACATCATCGATCAGCTCCCGCTCCCGGGCAAGCAGAAGTACATCCAGAACCTCAATGCCTCCGAGGAGCTCCTGTTCAGGAATCTCATCCTCGTGGATCTCCCGACCTACTGCGTCGATGCCATCGCGGCGGTCGGCCAGGATGTGCTGGATAAGTTCACCAAGGACATCCTCGAGATCGCCGAGCAG SEQ ID NO.2 (DNA sequence of the Cas12i2 variant Cas12i2 v1 functional component, 3162 bp): SEQ ID NO.3 (DNA sequence of the N-terminal bpNLS functional component, 54 bp): AAGCGGACGGCGGACGGGAGCGAGTTCGAGAGCCCGAAGAAGAAGAGGAAGGTG SEQ ID NO.4 (DNA sequence of the C-terminal bpNLS functional component, 54 bp): AAACGCACCGCCGACGGGTCCGAGTTTGAGTCCCCCAAAAAGAAGAGAAAAGTG SEQ ID NO.5 (DNA sequence of linker1 functional component, 45 bp): GGCGGCGGCGGATCTGGAGGAGGAGGAAGCGGCGGCGGCGGTTCT SEQ ID NO.6 (Amino acid sequence of 5′ exonuclease T5E, 290 aa): SKSWGKFIEEEEAEMASRRNLMIVDGTNLGFRFKHNNSKKPFASSYVSTIQSLAKSYSARTTIVLGDKGKSVFRLEHLPEYKGNRDEKYAQRTEEEKALDEQFFEYLKDAFELCKTTFPTFTIRGVEADDMAAYIVKLIGHLYDH VWLISTDGDWDTLLTDKVSRFSFTTRREYHLRDMYEHHNVDDVEQFISLKAIMGDLGDNIRGVEGIGAKRGYNIIREFGNVLDIIDQLPLPGKQKYIQNLNASEELLFRNLILVDLPTYCVDAIAAVGQDVLDKFTKDILEIAEQ SEQ ID NO.7 (Amino acid sequence of Cas12i2 variant Cas12i2 v1, 1054 aa): SEQ ID NO.8 (amino acid sequence of N-terminal bpNLS, 18 aa): KRTADGSEFESPKKKRKV SEQ ID NO.9 (Amino acid sequence of C-terminal bpNLS, 18 aa): KRTADGSEFESPKKKRKV SEQ ID NO.10 (amino acid sequence of linker1, 15 aa): GGGGSGGGGSGGGGS; The optimized synthetic bpNLS-T5E-linker1 and Cas12i2 v1-bpNLS were ligated by overlapping PCR to form a fully fused T5E-Cas12i2 v1, which was then cloned into the binary vector pYLCRISPR-Cas9Pubi-H (Ma et al., 2015, Molecular Plant, 8: 1274–1284) using the Gibson assembly method. Pst I and BamH Between I and II, the editor T5E-Cas12i2 v1 was obtained. Furthermore, bpNLS and Cas12i2 v1-bpNLS were combined via overlapping PCR to form a fully fused Cas12i2 v1, which was then cloned into the binary vector pYLCRISPR-Cas9Pubi-H. Pst I and BamH Between steps I and II, the editor Cas12i2 v1 was obtained as a control to examine the role of T5E in genome editing. The complete base sequence encoding the T5E-Cas12i2 v1 fusion protein and the complete amino acid sequence of the fusion protein T5E-Cas12i2 v1 are shown in SEQ ID NO.11 and SEQ ID NO.12, respectively: SEQ ID NO.11 (complete base sequence encoding the T5E-Cas12i2 v1 fusion protein, 4191 bp): SEQ ID NO.12 (Complete amino acid sequence of fusion protein T5E-Cas12i2 v1, 1396 aa): The specific construction method is as follows: The primers used in the construction process are shown in Table 1: Table 1. Basic Carrier Modifications for Cas12i2 v1 and T5E-Cas12i2 v1

[0042] (1) Using F-T5E-1 / R-T5E-1 (SEQ ID NO.13 and SEQ ID NO.14) primers, the synthesized bpNLS-T5E-linker1 plasmid was used as a template to amplify the bpNLS-T5E-linker1 fragment.

[0043] PCR system (15 µL): 2×PhantaMax Buffer 7.5 µL, 10 mM dNTPs Mix 0.35 µL, PhantaMax Polymerase 0.35 µL, bpNLS-T5E-linker1 plasmid 10 ng, 10 µM F-T5E-1 0.35 µL, 10 µM R-T5E-1 0.35 µL, ddH2O to 15 µL.

[0044] PCR program: pre-denaturation at 95℃ for 1 min, 28 PCR cycles (95℃ for 10 s, 56℃ for 15 s, 72℃ for 30 s), extension at 72℃ for 1 min.

[0045] (2) Using F-Cas12i2 v1-2 / R-Cas12i2 v1-2 (SEQ ID NO.15 and SEQ ID NO.16) primers, the synthesized Cas12i2 v1-bpNLS plasmid was used as a template to amplify the Cas12i2 v1-bpNLS fragment.

[0046] PCR system (15 µL): 2×PhantaMax Buffer 7.5 µL, 10 mM dNTPs Mix 0.35 µL, PhantaMax Polymerase 0.35 µL, Cas12i2 v1-bpNLS plasmid 10 ng, 10 µM F-Cas12i2 v1-2 0.35 µL, 10 µM R-Cas12i2 v1-2 0.35 µL, ddH2O to bring the total to 15 µL.

[0047] PCR program: pre-denaturation at 95℃ for 1 min, 28 PCR cycles (95℃ for 10 s, 56℃ for 15 s, 72℃ for 2 min), extension at 72℃ for 2 min.

[0048] (3) Using F-T5E-1 / R-Cas12i2 v1-2 (SEQ ID NO.13 and SEQ ID NO.16) primers, the bpNLS-T5E-linker1 and Cas12i2 v1-bpNLS fragments amplified in the first round were used as templates to amplify the bpNLS-T5E-linker1-Cas12i2 v1-bpNLS fusion DNA fragment.

[0049] PCR system (15 µL): 2×PhantaMax Buffer 7.5 µL, 10mM dNTPs Mix 0.35 µL, PhantaMax Polymerase 0.35 µL, 0.1 µL each of the first-round amplified bpNLS-T5E-linker1 and Cas12i2 v1-bpNLS fragments, 0.35 µL of 10 µM F-T5E-1, 0.35 µL of 10 µM R-Cas12i2 v1-2, and ddH2O to a final volume of 15 µL.

[0050] PCR program: pre-denaturation at 95℃ for 1 min, 28 PCR cycles (95℃ for 10 s, 56℃ for 15 s, 72℃ for 3 min), extension at 72℃ for 3 min.

[0051] (4) Using F-bpNLS-3 / R-bpNLS-3 (SEQ ID NO.17 and SEQ ID NO.18) primers, the synthesized Cas12i2 v1-bpNLS plasmid was used as a template to amplify the complete bpNLS-Cas12i2 v1-bpNLS fragment.

[0052] PCR system (50 µL): 2×PhantaMax Buffer 25 µL, 10mM dNTPs Mix 1.0 µL, PhantaMax Polymerase 1.0 µL, Cas12i2 v1-bpNLS plasmid 10 ng, 10 µM F-bpNLS-3 1.0 µL, 10 µM R-bpNLS-3 1.0 µL, ddH2O to 50 µL.

[0053] PCR program: pre-denaturation at 95℃ for 1 min, 28 PCR cycles (95℃ for 10 s, 56℃ for 15 s, 72℃ for 2 min), extension at 72℃ for 2 min.

[0054] (5) Using the Genstar purification kit, purify the PCR products of the amplified bpNLS-T5E-linker1-Cas12i2 v1-bpNLS and bpNLS-Cas12i2 v1-bpNLS fusion DNA fragments, respectively. Pst I and Bam The pYLCRISPR-Cas9Pubi-H plasmid was digested with HI enzyme.

[0055] Reaction system (10 µL) and procedure: 10x Faster digest buffer. Pst I 0.5 µL, Bam HI 0.5µL, pYLCRISPR-Cas9Pubi-H 300 ng, ddH2O to make up to 10 µL, react at 37℃ for 1 h, and recover the carrier skeleton from the gel.

[0056] The fused DNA fragment and vector were then ligated using a Gibson assembly reaction (NEB #E5510S).

[0057] Reaction system (10 µL) and procedure: 2x Mix 5 µL, bpNLS-T5E-linker1-Cas12i2 v1-bpNLS or bpNLS-Cas12i2 v1-bpNLS fragment 60 ng, gel-recovered carrier backbone 90 ng, ddH2O to make up to 10 µL, react at 50 °C for 50 min.

[0058] 1.5 µL of the Gibson ligation product was used to transform *E. coli* Top10 cells using electroporation. Single clones were screened and transformed on kanamycin-resistant (Kana) LB plates. Positive clones were sequenced to obtain the T5E-Cas12i2 v1 and Cas12i2 v1 basal vector plasmids (see [link to original text]). Figure 1 ).

[0059] Example 2. T5E-Cas12i2 v1 exhibits higher DNA cutting and editing efficiency and long fragment deletion efficiency. Twelve crRNA sequences were designed targeting 12 endogenous rice sites, and tRNA ribozyme sequences were added to the 5′ end of each crRNA, along with HDV ribozyme sequences at the 3′ end (see [link to crRNA sequence]). Figure 2 This helps the crRNA expression cassette be processed into a single crRNA in vivo. The crRNA expression cassette sequence was provided by Wuhan Genecreate Company. Figure 2 Sequences are synthesized directly. Using P... 35S-CmYLCV-sU6A composite promoter (Jiang et al., 2020, Genome Biology, 21: 257) drives crRNA expression. Gibson assembly is used to insert the crRNA expression cassette into the Cas12i2 v1 and T5E-Cas12i2 v1 binary vectors. Sgs Between I restriction enzyme sites. After transformation into rice, the target sites of the transgenic plants were sequenced to analyze the editing efficiency of Cas12i2 v1 and T5E-Cas12i2 v1.

[0060] The specific construction and transformation methods are as follows: The primers used in the construction process are shown in Table 2: Table 2 Primers for constructing crRNA expression cassettes

[0061] (1) Using F-crRNA / R-crRNA (SEQ ID NO.19 and SEQ ID NO.20) primers, the crRNA expression cassette fragment was amplified using the company's synthetic crRNA expression cassette plasmid as a template.

[0062] PCR system (50 µL): 2×PhantaMax Buffer 25 µL, 10mM dNTPs Mix 1.0 µL, PhantaMax Polymerase 1.0 µL, crRNA expression cassette plasmid 10 ng, 10 µM F-crRNA 1.0 µL, 10 µM MR-crRNA 1.0 µL, ddH2O to 50 µL.

[0063] PCR program: pre-denaturation at 95℃ for 1 min, 28 PCR cycles (95℃ for 10 s, 56℃ for 15 s, 72℃ for 1 min), extension at 72℃ for 1 min.

[0064] (2) Purify the PCR product of the amplified crRNA expression cassette using the Genstar purification kit. Sgs I digested the Cas12i2 v1 and T5E-Cas12i2 v1 plasmids with enzyme I.

[0065] Reaction system (10 µL) and procedure: 10x Faster digest buffer. Sgs I 1.0 µL, Cas12i2 v1 or T5E-Cas12i2 v1 plasmid 300 ng, ddH2O to make up to 10 µL, react at 37℃ for 1 h, and then recover the carrier backbone using gel.

[0066] The fused DNA fragment and vector were then ligated using a Gibson assembly reaction (NEB #E5510S).

[0067] Reaction system (10 µL) and procedure: 5 µL of 2x Mix, 60 ng of crRNA expression cassette fragment, 90 ng of gel-extracted vector backbone, and ddH2O to bring the total to 10 µL. Incubate at 50 °C for 50 min.

[0068] 1.5 µL of the Gibson ligation product was used to transform *E. coli* DH10B via electroporation. Single clones were screened on kanamycin-resistant (Kana) LB plates. Positive clones were sequenced using SP-L1 / SP-R (SEQ ID NO.21 and SEQ ID NO.22) primers to obtain the T5E-Cas12i2 v1 and Cas12i2 v1 binary vector plasmids carrying the crRNA expression cassette, respectively.

[0069] (3) The T5E-Cas12i2 v1 and Cas12i2 v1 binary vector plasmids carrying crRNA expression cassettes were transformed into Agrobacterium EHA105 and infected rice callus tissue. Leaf DNA was extracted from T0 generation transformed plants as templates to amplify the DNA fragments of the editing target sites. After recovery, high-throughput sequencing was performed. The editing efficiency of Cas12i2v1 and T5E-Cas12i2 v1 was statistically compared by comparing the sequencing results with the reference sequence. The results showed that the average editing efficiency of T5E-Cas12i2 v1 was higher (45.7%), while the average editing efficiency of Cas12i2 v1 was only 12.1% ( Figure 1 B, 1C). Furthermore, these editors are more efficient at editing targets with 5′-TTT (47.5% efficiency) and 5′-TTG PAM (39.9% efficiency) compared to targets with 5′-TTA (16.5% efficiency) and 5′-TTCPAM (7.8% efficiency). Figure 1 (B, 1C) indicates that the plant Cas12i2 v1 editor has a preference for targeting 5′-TTT and 5′-TTG PAM sites.

[0070] Traditional Cas12 effectors exhibit staggered DNA cleavage activity, tending to produce small DNA deletions and not suitable for manipulating long DNA fragments or cis elements. To investigate whether Cas12i2 v1, fused with the 5′ exonuclease T5E, can increase the size of deleted DNA fragments, this invention statistically analyzed the abilities of Cas12i2 v1 and T5E-Cas12i2 v1 in generating DNA fragment deletion sizes at 12 target sites. Taking two representative sites, TS4 and TS11, as examples, the analysis results showed that Cas12i2 v1 mainly produces small deletions, while T5E-Cas12i2 v1 can produce longer deletions exceeding 100 bp in length (see...). Figure 3 Further statistical analysis of the resulting DNA fragment deletion patterns revealed that deletions produced by Cas12i2 v1 were mainly concentrated in the 9–20 bp length range (89.6%), while T5E-Cas12i2 v1 primarily produced deletions in the 21–40 bp length range (46.6%). The probabilities of longer fragment deletions were 81–100 bp (4.5%) and 101–126 bp (2.2%), respectively (see [link to relevant documentation]). Figure 4 This indicates that the fusion of the 5′ exonuclease T5E significantly enhances the ability of Cas12i2 v1 to generate long-fragment deletions.

[0071] Example 3. Precise improvement of rice traits using T5E-Cas12i2 v1 manipulator large rotary element. Miniature inverted-repeat transposable elements (MITEs) are compact, non-autonomous DNA transposons, typically 100–800 bp in size, widely distributed throughout plant genomes. They play a role in regulating gene expression and translation levels, making them ideal target sites for crop genetic improvement. This invention uses the T5E-Cas12i2 v1 editor to manipulate MITEs in endogenous rice genes to demonstrate the application of T5E-Cas12i2 v1 in crop genetic improvement. Rice OsDOG Encoding the A20 / AN1 zinc finger protein, it negatively regulates gibberellin-mediated rice cell elongation. OsDOG At a position 929 bp upstream of the start codon, there exists a 351-bp long... PIF / Harbinger Superfamily MITE (see) Figure 5 A). Utilizing T5E-Cas12i2 v1 and a pair of adjacent OsDOG crRNA flanking MITE, for OsDOGMITE deletion was precisely performed, resulting in 30 T0 transgenic plants (Japonica rice Zhonghua 11 background, ZH11). PCR was used to detect MITE deletion in the T2 transgenic lines, identifying two lines, L7 and L17, with homozygous MITE deletion (see...). Figure 5 The deletion fragment was marked with an asterisk (B), and the boundary sequence of the deleted fragment was confirmed by Sanger sequencing (see [link]). Figure 5 C).

[0072] The seedling heights of wild-type ZH11, a non-MITE deletion line L3 (as a negative control), and two MITE homozygous deletion lines L7 and L17 were analyzed. After seed germination, these lines were cultured in hydroponic containers under the conditions of 28°C for 12 hours of light / 28°C for 12 hours of darkness. After 14 days of culture, seedling heights were measured. The results showed that compared to wild-type ZH11, the seedling heights of the MITE homozygous deletion lines L7 and L17 were significantly decreased, exhibiting a clear dwarfing phenotype. The seedling height of the non-MITE deletion line L3, however, showed no significant change (see...). Figure 6 A). Further measurements of plant height revealed that the average plant height of L7 seedlings was 0.68–0.75 times lower than that of wild-type ZH11 (see [reference]). Figure 6 B). Endogenous factors in plants OsDOG The expression levels were detected, and endogenous expression was found in the L7 and L17 strains. OsDOG Expression levels were significantly improved (see Figure 6 C), indicating that it is mediated by T5E-Cas12i2 v1 OsDOG The removal of MITE lifted the restrictions on internal sources. OsDOG The expression of inhibition, OsDOG Upregulation of expression levels led to a decrease in the height of rice seedlings.

[0073] In summary, the novel, highly efficient plant T5E-Cas12i2 v1 editor developed in this invention has advantages over traditional CRISPR-Cas9 editors, including high efficiency, targeting of the TTN-PAM site, and the ability to produce site-specific large fragment deletions. Furthermore, the fusion of the 5′ exonuclease T5E significantly improves the editor's editing efficiency and its ability to delete long DNA fragments and cis-elements, enabling its widespread application in crop gene function screening, editing regulatory elements, precise regulation of gene expression levels, and molecular breeding.

[0074] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0075] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0076] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A fusion protein T5E-Cas12i2 v1, a 5′ exonuclease and CRISPR-Cas12i2 variant for plant gene editing, characterized in that, From the N-terminus to the C-terminus, it contains, in sequence, a 5′ exonuclease T5E and a Cas12i2 enzyme variant Cas12i2 v1, the amino acid sequence of which is shown in SEQ ID NO.6 and the sequence of which is shown in SEQ ID NO.

7.

2. The 5′ exonuclease and CRISPR-Cas12i2 variant fusion protein T5E-Cas12i2v1 according to claim 1, characterized in that, It also includes two core localization signals respectively fused to the N-terminus and C-terminus of the T5E-Cas12i2 v1; and / or It also contains a flexible linker sequence for the fusion protein linking the 5′ exonuclease to the Cas12i2 v1 variant; Preferably, the nuclear localization signal is bpNLS, whose amino acid sequences at the N-terminus and C-terminus of the fusion protein are shown in SEQ ID NO. 8 and SEQ ID NO. 9, respectively; and / or The flexible linker sequence of the fusion protein is linker1, and its amino acid sequence is shown in SEQ ID NO.

10.

3. The 5′ exonuclease and CRISPR-Cas12i2 variant fusion protein T5E-Cas12i2v1 according to claim 1, characterized in that, From the N-terminus to the C-terminus, it contains, in sequence, the nuclear localization signal bpNLS1, the 5′ exonuclease T5E, the flexible linker sequence linker1, the Cas12i2 enzyme variant Cas12i2 v1, and the nuclear localization signal bpNLS2, and its full amino acid sequence is shown in SEQ ID NO.

12.

4. A polynucleotide sequence, characterized in that, The polynucleotide sequence encoding the 5′ exonuclease and CRISPR-Cas12i2 variant fusion protein T5E-Cas12i2 v1 as described in any one of claims 1 to 3; Preferably, the nucleotide sequence encoding the 5′ exonuclease T5E is as shown in SEQ ID NO.1, and the nucleotide sequence encoding the Cas12i2 enzyme variant Cas12i2 v1 is as shown in SEQ ID NO.2; and / or The nuclear localization signals encoding the N-terminus and C-terminus of the fusion protein are bpNLS, and their nucleotide sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively; and / or The nucleotide sequence encoding the flexible linker sequence connecting the 5′ exonuclease T5E to the Cas12i2 v1 variant is shown in SEQ ID NO. 5; Preferably, the polynucleotide sequence is as shown in SEQ ID NO.

11.

5. A plasmid vector containing the polynucleotide sequence of claim 4.

6. The use of the 5′ exonuclease and CRISPR-Cas12i2 variant fusion protein T5E-Cas12i2 v1 according to any one of claims 1 to 3, or the polynucleotide sequence according to claim 4, or the plasmid vector according to claim 5, in the preparation of a plant T5E-Cas12i2 v1 editor.

7. A plant T5E-Cas12i2 v1 editor, characterized in that, It is obtained by constructing the polynucleotide sequence encoding the 5′ exonuclease and CRISPR-Cas12i2 variant fusion protein T5E-Cas12i2 v1 as described in any one of claims 1 to 4 into a plant transformation vector.

8. The Plant T5E-Cas12i2 v1 editor according to claim 7, characterized in that, The plant transformation vector is a binary vector pYLCRISPR-Cas9Pubi-H.

9. The method for constructing the Plant T5E-Cas12i2 v1 editor according to claim 8, characterized in that, First, the complete fusion DNA sequence encoding the fusion protein T5E-Cas12i2 v1 of the 5′ exonuclease and the CRISPR-Cas12i2 variant was prepared, and then assembled between Pst I and BamH I of the binary vector pYLCRISPR-Cas9Pubi-H to obtain the editor T5E-Cas12i2v1.

10. The application of the plant T5E-Cas12i2 v1 editor of claim 8 or 9 in plant genome editing, preferably, the plant is rice.

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