Methods to increase guided editing efficiency in plants

Abstract

This invention relates to a method for increasing guided editing efficiency in plants, and more specifically, to a recombinant vector for guided editing in plants with increased correction efficiency and its use therein, the recombinant vector comprising a virus-based replicon comprising: a prime editing guide RNA (pegRNA) expression cassette regulated by a U6 composite promoter; and a prime editor expression cassette.

Description

Technical Field

[0001] This invention relates to a method for increasing the efficiency of prime editing in plants. Background Technology

[0002] Prime editing, a CRISPR-Cas9 tool initially described by Anzalone et al. in 2019 (Nature 2019, 576, 149-157), is reported to not induce double-strand breaks (DSBs) and can accurately correct all types of point mutations, insertions, and deletions, with the potential to recover approximately 89% of human pathogenic genetic variations.

[0003] Guided editing technology requires two main structural elements: first, a prime editor (PE) protein composed of reverse transcriptase fused to the nickase Cas9 (nCas9); and second, a prime editing guide RNA (pegRNA) that guides the PE to the target location and functions as a template for the intended correction. The pegRNA consists of a primer binding site (PBS) that acts as a docking site for the reverse transcriptase and a reverse transcription template (RTT) containing the intended correction information.

[0004] Following the initial reports of PE by Ansalon et al., there have been reports of improvements and applications of PE in various organisms. Recently, all aspects of the original PE structural elements have been modified and new properties added, continuously improving the efficiency of guided editing. However, in plants, the application of PE shows inconsistencies across different loci and species; in particular, much effort is still needed to improve the efficiency of guided editing in dicotyledonous plants.

[0005] Therefore, the present invention will provide a method for improving the efficiency of guided editing in plants, especially in dicotyledonous plants.

[0006] On the other hand, Korean Patent Publication No. 2022-0112698 discloses a "composition for guided editing with improved correction efficiency", and Korean Patent Publication No. 2023-0075420 discloses "guided editing using HIV reverse transcriptase and Cas9 or its variants", but there is no record of the "method for increasing guided editing efficiency in plants" of the present invention. Summary of the Invention

[0007] (a) Technical problems to be solved To increase the efficiency of prime editor in plants, this invention experiments with various combinations of prime editor constituent proteins, types of transcriptional regulators (promoters, terminators) of the prime editor constituent proteins and / or pegRNAs, transduction systems, and other conditions, and proposes an optimized combination to provide a method that can increase prime editor efficiency in plants.

[0008] (II) Technical Solution To address the aforementioned technical problem, the present invention provides a recombinant vector for plant-based guided editing with increased correction efficiency, comprising a virus-based replicon, wherein the virus-based replicon includes: a guide RNA expression cassette regulated by a U6 composite promoter; and a guide editor expression cassette.

[0009] Furthermore, the present invention provides a method for improving the efficiency of guided editing of plant cells, including the step of transforming plant cells using the recombinant vector.

[0010] Furthermore, the present invention provides a composition for improving the efficiency of guided editing correction in plants, comprising the recombinant vector as an active ingredient.

[0011] (III) Beneficial Effects When using the method of the present invention, the efficiency of guided editing of plants, especially dicotyledonous plants, can be improved. The method of the present invention can be effectively utilized in the field of new variety breeding based on precision gene editing technology. Attached Figure Description

[0012] Figure 1This is a schematic diagram of the Guided Editor (PE) expression box used in this invention. p35S: CaMV 35S promoter; nCas9max: mutant SpCas9 (R221K, N394K, D840A); nCas9: mutant SpCas9 (D840A); MMLV-RT: mutant MMLV reverse transcriptase (D200N, T306K, W313F, T330P, L603W); MMLV-RT-△RnaseH: domain-truncated mutant MMLV reverse transcriptase (D200N, T306K, W313F, T330P, L603W); MHL1dn: truncated MLH1D754-756 (lacking endonuclease domain); NC: viral nucleocapsid protein. protein), EURb7: EU + Rb7 dual terminator, MCP-MMLV-RT: MS2 capsid protein fused MMLV-RT (MCP was cloned from pREDIT_MCP-RecT, MMLV RT is an atobacco codon-optimized MMLV RT from the PE2 system).

[0013] Figure 2 and Figure 3 This is a schematic diagram of the recombinant vector used to guide editing in an embodiment of the present invention. Figure 2 This is a vector that regulates pegRNA expression via the U6-26 core promoter. Figure 3 This is a vector for regulating pegRNA expression via the U6 complex promoter. Supp.gRNA indicates expression regulated via a promoter used for... ALS1 The gRNA that guides the editing reaction binds to the adjacent position of the pegRNA, which is composed of the base sequence of SEQ ID NO: 258. pegR1.6-MS2 is composed of the base sequence of SEQ ID NO: 257. pegR1.8 is formed by linking the gRNA and the base sequence of RTT+PBS to both ends of the Cas9 gRNA scaffold sequence (equivalent to the base sequence from the 92nd to the 177th base in SEQ ID NO: 258) (Table 1).

[0014] Figure 4 To utilize Figure 2 and Figure 3 Analysis results of the correction efficiency of the guided editing of the recombinant vector. p < 0.05 p < 0.0002 p < 0.0001.

[0015] Figure 5 To analyze the expected frequency and efficiency of guided editing in redifferentiated plants. The y-axis values ​​in the graph represent the results in the transformant (from the plant using...) Figure 2 or Figure 3 The redifferentiated plants from cotyledonary explants transformed by the vector, i.e., , represents the frequency (%) of guided editing by Sanger sequencing analysis at the target location in the PE0 event. The values ​​at the top of each graph represent the expected efficiency of guided editing in the plant stage, calculated by dividing the number of transformants corresponding to the expected guided editing by the total number of transformants analyzed.

[0016] Figure 6 Polymerase chain reaction (PCR) results used to confirm the presence or absence of replicons and T-DNA in a subset of PE0 event plants.

[0017] Figure 7 This is a schematic diagram of recombinant vectors used to compare the editing efficiency guided by Agrobacterium strains.

[0018] Figure 8 The results show the efficiency of editing guided by Agrobacterium strain analysis. p < 0.05.

[0019] Figure 9 To analyze the expected frequency and efficiency of guided editing in redifferentiated plants during guided editing experiments using Agrobacterium strains.

[0020] Figure 10 This is a schematic diagram of recombinant vectors used to compare the efficiency of guided editing with T-DNA-based or replicon-based recombinant vectors. pEL2B-3: Golden Gate endlinker (position 3).

[0021] Figure 11 Results guided by T-DNA or replicon-based recombinant vector analysis improve editing efficiency. p < 0.0002 p < 0.0001.

[0022] Figure 12 This is a schematic diagram of a recombinant carrier used to compare the efficiency of guided editing under different temperature conditions.

[0023] Figure 13 This is the result of the guided editing efficiency analysis based on temperature conditions. p < 0.0021 p < 0.0002 p < 0.0001.

[0024] Figure 14 This study aims to analyze the expected frequency and efficiency of guided editing in plant samples during guided editing experiments conducted under specific temperature conditions.

[0025] Figure 15 This is a schematic diagram of a recombinant vector used to compare the efficiency of guided editing for multiple locations and loci. The npegRNA consists of tRNA (Gly) - gRNA of the target gene - altered (F+E) SpCas9 gRNA backbone - RTT-tevopreQ1-HDV sequence of the target gene, and is composed of the base sequence of SEQ ID NO: 259.

[0026] Figure 16 The results show the efficiency analysis of guided editing for various target locations and positions on a tomato plant.

[0027] Figure 17 A schematic diagram of a bootstrap editor expression box containing other terminators.

[0028] Figure 18 This is a schematic diagram of a recombinant carrier used to compare the efficiency of guided editing with the termination sub-types of guided editor expression boxes.

[0029] Figure 19 The results of the guided editing efficiency analysis are based on the termination sub-category analysis of the guided editor's expression box.

[0030] Figure 20 This is a schematic diagram of a bootstrap editor expression box that contains other starters.

[0031] Figure 21 This is a schematic diagram of a recombinant carrier used to compare the efficiency of guided editing with different types of promoters in guided editor expression boxes.

[0032] Figure 22 The results of analyzing the startup types of the guided editor expression box are used to guide the editing efficiency.

[0033] Figure 23 This is a schematic diagram illustrating the different types and sequences of RNA chaperone proteins.

[0034] Figure 24 This is a schematic diagram of a recombinant vector used to compare the efficiency of guided editing based on the presence, type, and sequence of RNA chaperone proteins.

[0035] Figure 25 The results show that the efficiency of editing is guided by the presence, type, and sequence of RNA chaperone proteins.

[0036] Figure 26 In order to be in Figure 25 The results analyze the editing type of the target location.

[0037] Figure 27 This is a schematic diagram of the guide editor expression cassette PE6, which contains PE2max and other nickases Cas9 and / or reverse transcriptases. The coding sequences of the guide editors PE6c, PE6c-NC, PE6d, PE6d-NC, PE6ec, PE6ec-NC, PE6fc, PE6fc-NC, PE6gc, and PE6gc-NC are composed of the base sequences of SEQ ID NO: 260 to SEQ ID NO: 269, respectively. nCas9max: mutant SpCas9 (R221K, N394K, D840A), SpG-nCas9max: mutant SpCas9 (R221K, N394K, H840A, D1135L, S1136W, G1218K, E1219Q, R1335Q, T1337R), evoTf1-RT: mutant Tf1 retrotransposon reverse transcriptase (P70T, G72V, S87G, M102I, K106R, K118R, I128V, L158Q, F269L, A363V, K413E, S492N, S188K, I260L, S297Q, R288Q).

[0038] Figure 28 This is a schematic diagram of a recombinant carrier used to compare the efficiency of guided editing with different types of expression boxes in the PE6 guided editor.

[0039] Figure 29This study analyzes the editing efficiency of the editor based on the types of expression boxes used in the PE6-guided editor. PE2max is used as a comparative control group.

[0040] Figure 30 The results were used to compare the boot editing efficiency of the PE2max-NC boot editor and the PE6c boot editor.

[0041] Figure 31 This is a schematic diagram of a recombinant vector targeting six tomato genes, used to test the improved editing efficiency of the PE6c guide editor.

[0042] Figure 32 To analyze the guided editing efficiency of the PE6c guided editor targeting six tomato genes, PE2max was used as a control group.

[0043] Figure 33 For SlHKT1;2 A schematic diagram of a gene-targeting recombinant vector containing multiple PE6 guide editor expression cassettes.

[0044] Figure 34 To analyze using various PE6 boot editors SlHKT1;2 Results of gene-targeted guided editing efficiency.

[0045] Figure 35 For use with PE6c, PE6c-NC, or PE6ec-NC boot editor expression boxes SlOR , SlCAB13 and SlCENH3 ( sub (A schematic diagram of a gene-targeting recombinant vector.)

[0046] Figure 36 To analyze the efficiency of the PE6c, PE6c-NC, and PE6ec-NC guided editors in guiding the editing of three tomato genes, PE2max was used as a control group.

[0047] Figure 37 To analyze the Arabidopsis thaliana plant body as the subject of study AtPDS3 The results of the editing efficiency of the PE2max-NC, PE6c, or PE6c-NC guided editors targeting genes are shown in the schematic diagram of the recombinant vectors used and the chart of guided editing efficiency analysis.

[0048] Figure 38 To analyze the Arabidopsis thaliana plant body as the subject of study AtCENH3 , AtOR and AtALS The results of the editing efficiency of the PE2max-NC or PE6c-NC guided editors targeting genes are shown in the schematic diagram of the recombinant vectors used and the guided editing efficiency analysis table. Detailed Implementation

[0049] To achieve the objectives of this invention, the present invention provides a recombinant vector for plant guided editing with increased correction efficiency, comprising a virus-based replicon, said virus-based replicon comprising: a guide RNA expression cassette regulated by a U6 complex promoter; and a guide editor expression cassette.

[0050] A guide editor is a type of gene-editing system based on CRISPR / Cas9, capable of introducing gene changes by cutting only single-stranded DNA without cutting the double-stranded DNA. The guide editor may contain a nickase Cas-reverse transcriptase (RT) fusion protein and guide RNA (pegRNA). To improve the efficiency of the guided editing, additional domains or proteins may be included in the fusion protein.

[0051] The term "pegRNA" in this invention includes a guide sequence or spacer sequence for recognizing a target sequence, a trans-activating crispr RNA backbone sequence, a primer binding site (PBS) required for initiating reverse transcription, and a reverse transcription template (RTT) containing the desired gene change. Furthermore, the pegRNA may also include variants such as tevopreQ1 fusion that can enhance the stability of the pegRNA.

[0052] In the pegRNA, the guide sequence refers to a sequence within the guide RNA at the designated target site, including sequences that are all or part of the target sequence complementary. The guide sequence is any nucleotide sequence complementary to the target polynucleotide sequence that hybridizes with the target DNA sequence and is sufficient to induce sequence-specific binding of the gene-editing complex to the target DNA sequence.

[0053] In the recombinant vector of the present invention, the U6 complex promoter may be composed of the base sequence of SEQ ID NO: 10, but is not limited thereto.

[0054] In this invention, the term "replicon" refers to a self-controlled replication unit, which is a continuous DNA molecule that replicates sequentially from a specific location within the molecule. Plasmids, viral DNA, and bacterial chromosomes are all examples of a single replication unit.

[0055] In the recombinant vector of the present invention, the virus-based replicon can be, but is not limited to, based on Bean Yellow Dwarf Virus (BeYDV), Maize Streak Virus (MSV), Tobacco Mosaic Virus (TMV), Cauliflower Mosaic Virus (CaMV), Tobacco Ringspot Virus (TRSV), Tobacco Etch Virus (TEV), Potato Spindle Tuber Viroid (PSTVd), Cucumber Mosaic Virus (CMV), Papaya Ringspot Virus (PRSV), Tobacco Streak Virus (TSV), Pea Enation Mosaic Virus (PEMV), Potato Virus X (PVX), Potato Virus Y (PVY), and Potato Leafroll Virus. Virus, PLRV, Cowpea mosaic virus (CPMV), Bean common mosaic virus (BCMV), Beet curly topvirus (BCTV), Alfalfa mosaic virus (AMV), Tomato spotted wilt virus (TSWV), Beet yellows virus (BYV), Cucumber green mottle mosaic virus (CGMMV), Turnip yellowmosaic virus (TYMV), Tobacco vein mottling virus (TVMV), Soybean mosaic virus (SMV), Rice tungro bacilliform virus (RTBV), Rice stripe virus (RSV), Rice dwarf virus (RSV).The virus may contain replicons of RDV, Maize chlorotic mottle virus (MCMV), Maize dwarf mosaic virus (MDMV), Maize chlorotic dwarf virus (MCDV), Barley stripe mosaic virus (BSMV), Wheat streak mosaic virus (WSMV), Wheat dwarf virus (WDV), or Wheat streak mosaic virus (WSMV), preferably, but not limited to, replicons of geminiviruses such as BeYDV or MSV.

[0056] The replicon can be sequentially linked with a long intergenic region (LIR), a promoter, a Rep / RepA protein-coding sequence, a terminator, a short intergenic region (SIR), a multiple cloning site (MCS) for inserting the desired exogenous gene, the SIR, and the LIR, but is not limited to these. The viral LIR functions as an origin of replication and a promoter, while the SIR functions as a terminator. The virus-based replicon vector can be referenced from the inventors' previous work (Korean Patent No. 2074744).

[0057] In this invention, a virus-based replicon is carried on T-DNA. After being injected into a plant by Agrobacterium, a circular replicon is prepared by rolling circle replication and the carried recombinant sequence is expressed.

[0058] In the recombinant vector of the present invention, the replicon may further include a guide editor expression box and a screening marker expression box.

[0059] In a recombinant vector of one embodiment of the present invention, the guide editor may be: a fusion protein 1 consisting of Cas9 (CRISPR-associated protein 9) protein, Moloney murine leukemia virus reverse transcriptase (MMLV) reverse transcriptase, and nucleocapsid protein; a fusion protein 2 consisting of Cas9 protein and Tf1 retrotransposon reverse transcriptase protein; or a fusion protein 3 consisting of Cas9 protein, Tf1 retrotransposon reverse transcriptase, and nucleocapsid protein. Fusion protein 1 may be encoded by the base sequence of SEQ ID NO: 255, fusion protein 2 may be encoded by the base sequence of SEQ ID NO: 260 or SEQ ID NO: 264, and fusion protein 3 may be encoded by the base sequence of SEQ ID NO: 261 or SEQ ID NO: 265, but is not limited thereto.

[0060] In one embodiment of the present invention, the boot editor encoded by the base sequence of SEQ ID NO: 255 is PE2max-NC, the boot editor encoded by the base sequence of SEQ ID NO: 260 is PE6c, the boot editor encoded by the base sequence of SEQ ID NO: 264 is PE6ec, the boot editor encoded by the base sequence of SEQ ID NO: 261 is PE6c-NC, and the boot editor encoded by the base sequence of SEQ ID NO: 265 is PE6ec-NC (see reference). Figure 23 and Figure 27 ).

[0061] Furthermore, in one embodiment of the present invention, the cleavage enzyme Cas9 protein of PE2max-NC, PE6c, and PE6c-NC guided editor can be SpCas9 (Streptococcus pyogenes) with R221K, N394K, and D840A mutations. Streptococcus pyogenes The cleavage enzyme Cas9 protein of the PE6ec and PE6ec-NC guide editor can be SpCas9 with K775R, H840A and K918A mutations, but is not limited thereto. It can also be the cleavage enzyme Cas9 protein (SpG-nCas9max, encoded by the base sequence of SEQ ID NO: 278) with R221K, N394K, H840A, D1135L, S1136W, G1218K, E1219Q, R1335Q and T1337R mutations.

[0062] Furthermore, the MMLV reverse transcriptase of the PE2max-NC guide editor may have D200N, T306K, W313F, T330P and L603W mutations, and the Tf1 reverse transcriptase of the PE6c, PE6ec, PE6c-NC and PE6ec-NC guide editors may have P70T, G72V, S87G, M102I, K106R, K118R, I128V, L158Q, F269L, A363V, K413E, S492N, S188K, I260L, S297Q and R288Q mutations, but is not limited to these.

[0063] The expression box of the boot editor in one embodiment of the present invention can be adjusted by the CaMV 35S promoter and the EURb7 terminator (EU+Rb7 dual terminator), but is not limited thereto.

[0064] The guided editor of the present invention is characterized in that it is more efficient in guiding editing within a plant compared to guided editors composed of combinations of other elements.

[0065] Furthermore, in the recombinant vector of the present invention, the selection marker is used to screen cells transformed using the vector, and various markers that confer selective phenotypes such as drug resistance, auxotrophic effects, anticytotoxic agents, or expression of surface proteins can be used. Only cells expressing the selection marker survive in an environment treated with a selective agent; therefore, transformed cells can be screened.

[0066] In this invention, the term "recombinant" refers to a cell that replicates a xenogeneic nucleic acid, or expresses said nucleic acid or a protein encoded by a peptide, a xenogeneic peptide, or a xenogeneic nucleic acid. Recombinant cells can express genes or gene fragments not found in the natural form of said cell in either a sense or antisense form. Furthermore, recombinant cells can express genes found in the natural state of the cell, but said genes are modified genes reintroduced into the cell through artificial means.

[0067] Furthermore, the term "vector" is used when referring to DNA fragments or nucleic acid molecules transported into cells. Vectors can replicate DNA and reproduce independently within the host cell. The terms "transporter" and "vector" are often used interchangeably.

[0068] The term "expression cassette" in this invention refers to a sequence that regulates the expression of one or more genes, for example, a nucleic acid sequence containing any combination of multiple cis-acting transcriptional regulatory elements. The expression cassette of this invention comprises the following three main elements: i) a promoter; ii) a second polynucleotide operatively linked to the promoter, which, when the expression cassette is introduced into a cell, may also be referred to as a "coding polynucleotide" or "coding sequence" (also called a coding gene) that indicates its transcription by the promoter; and iii) a terminator polynucleotide (also called a transcription termination factor) indicating the end of transcription, located directly downstream of the second polynucleotide.

[0069] In this invention, the term "promoter" refers to the upstream region of DNA starting from a structural gene, and is a DNA molecule that binds to RNA polymerase to initiate transcription. "Plant promoter" refers to a promoter that can initiate transcription in plant cells. "Constitutive promoter" refers to a promoter that is active under most environmental conditions and in most developmental states or cell differentiation. The selection of transformants can be achieved at various stages and through various tissues; therefore, constitutive promoters are preferred in this invention. Thus, the selection of a constitutive promoter does not limit its functionality.

[0070] In this invention, the promoter is a promoter suitable for transformation. Preferably, it can be the CaMV 35S promoter, U6-26 core promoter, U6 complex promoter, actin promoter, ubiquitin promoter, pEMU promoter, MAS promoter, or histone promoter, but it is not limited to these.

[0071] In this invention, the terminator can be a common terminator, such as EURb7 (EU+Rb7 double terminator), t3T (EU+35S+Rb7 triple terminator), nopaline synthase (NOS) terminator, rice α-amylase RAmy1 A terminator, phaseolin terminator, terminator of Agrobacterium tumefaciens octopine synthase gene, etc., but is not limited to these.

[0072] Furthermore, the present invention provides a method for improving the efficiency of guided editing of plant cells, including the step of transforming plant cells using the recombinant vector of the present invention.

[0073] In the plant-guided editing efficiency enhancement method of the present invention, the recombinant vector is as described above.

[0074] Plant transformation refers to any method of transferring DNA into a plant. Such transformation methods do not necessarily involve regeneration and / or tissue culture. Currently, plant species transformation is a common procedure for both dicotyledonous and monocotyledonous plants. In principle, any transformation method can be used when introducing the hybrid DNA of this invention into suitable progenitor cells. Methods include the calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., 1987, Plant Mol. Biol. 8, 363-373), electroporation of protoplasts (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microinjection of plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185), particle bombardment of various plant elements (DNA or RNA-encoded) (Klein TM et al., 1987, Nature 327, 70), and transformation of Agrobacterium tumefaciens through plant infiltration, mature pollen, or microspores. Agrobacterium tumefaciens In gene transfer mediated by Agrobacterium, appropriate methods are selected from those such as transfection with (incomplete) viruses (EP 0 301316). Preferred methods of the present invention include Agrobacterium-mediated DNA transfer.

[0075] In one embodiment of the present invention, the transformation may be mediated by a strain having a genetic background of Agrobacterium tumefaciens EHA105 or EHA105 superagro ver.2, but is not limited thereto.

[0076] The transformation mediated by Agrobacterium tumefaciens can be carried out by treating plant tissues at a temperature of 30-35°C after co-culturing with Agrobacterium tumefaciens, preferably at a temperature of 30-32°C, more preferably at a temperature of 31°C, but not limited thereto.

[0077] The "plant cells" used in plant transformation can be any plant cell. Plant cells can be cultured cells, cultured tissues, cultured organs, or the entire plant. "Plant tissues" include differentiated or undifferentiated plant tissues, such as roots, stems, leaves, pollen, seeds, female tissues, and various cell forms used in culture, namely, single cells, protoplasts, buds, and callus. Plant tissues can be in plant (in planta), organ culture, tissue culture, or cell culture.

[0078] In the method of this invention, the transformed plant cells should redifferentiate into a whole plant. The method for redifferentiating the transformed plant cells into a transformed plant can utilize any method known in the art to which this invention pertains. Techniques for redifferentiating mature plants from numerous species by culturing callus or protoplasts are well known in the art to which this invention pertains.

[0079] Furthermore, the present invention provides a composition for improving the efficiency of guided editing correction in plants, comprising the recombinant vector of the present invention as an active ingredient.

[0080] In the compositions of the present invention, the recombinant vector is as described above. The compositions of the present invention are characterized by improved efficiency of guided editing at target sites in the plant.

[0081] The present invention will now be described in detail through embodiments. However, the following embodiments are merely illustrative of the invention, and the scope of the invention is not limited to these embodiments.

[0082] Experimental methods 1. Combination of PE protein elements and plasmid construction To produce the PE protein used in this invention, PE2max and PE4max, nCas9 (H840A) of ePEmax1, nCas9 of ePEmax2 and ePEmax3, and eRT were cloned from plasmid pCMV-PEmax-P2A-hMLH1dn (Addgene #174828). The nCas9 of PPE-NC-v1 was cloned from the nCas9-PPE plasmid (Addgene #140445), and NC and RT were cloned from the pH-ePPE plasmid (Addgene #183097). ePPE was cloned from the pH-ePPE plasmid, and the NC sequence used in ePEmax1, ePEmax2, ePEmax3, and PE2max-NC was cloned from the pH-ePPE plasmid.

[0083] In the case of the basic PE expression cassette, transcription was controlled using the CaMV 35S promoter (p35S) (Addgene #50267) and the EU+Rb7 double terminator (EURB7) (Diamos and Mason, 2018, Plant Biotechnol J.16:1971-1982). For experiments assessing the effect of the promoter, multiple promoters (p35S, the double CaMV 35S promoter (p2x35S) (Addgene #50269), and tomato EF1α (PSLEF1α)) were used in conjunction with the EU+35S+Rb7 triple terminator (t3T) (Niu et al., Plant Biotechnol J.2023, 21:5-7). To assess the effect of the terminator on PE efficiency, p35S was combined with multiple terminators (t35S, tNOS, EURB7, t3T).

[0084] Modified epigRNAs were designed by introducing fundamental changes into the epigRNA backbone within a modified SpCas9 backbone (Nelson et al., Nat Biotechnol. 2022, 40:402-410). To drive pegRNA transcription, either the pU6-26 core promoter or the U6 complex promoter was used (Jiang et al., GenomeBiol. 2020, 21:257). Table 1 details the edited loci, gRNA, and RTT information. After cloning the pegRNA into an expression cassette via PCR amplification, it was then bound to a binary vector using golden gate assembly technology.

[0085] In the case of T-DNA-based PE tools, the expression cassettes of the selection marker (NPTⅡ, Addgene #51144), PE protein, and pegRNA were cloned into the pAGM4723 plasmid (Addgene #48015). In the case of using a geminivirus replication system, the expression cassettes were cloned into the previously reported pLSL.R.Ly vector (Vu et al., Plant Biotechnol J. 2020, 18: 2133-2143). All biological components [pNOS::NptⅡ::tOCS (from pICSL11024, AddgenePlasmid #51144), PE2max (SEQ ID NO: 1), PE4max (SEQ ID NO: 2), PPE-NC-v1 (SEQ ID NO: 3), ePPE (SEQ ID NO: 4), ePEmax1 (SEQ ID NO: 5), ePEmax2 (SEQ ID NO: 6), PE2max-NC (SEQ ID NO: 255), ePEmax3 (SEQ ID NO: 256), 35S promoter (SEQ ID NO: 7), U6-26 core promoter (SEQ ID NO: 8), U6 composite promoter (SEQ ID NO: 9), EU + Rb7 dual terminator (EURb7; SEQ ID NO: 10), EU + 35S + Rb7 triple terminator (t3T; SEQ ID NO: 11)] were modified to Moclo (Weber et al., 2011, PLoS) One.6(2):e16765) Level 0 plasmid (domesticated) was assembled into a PE protein combination and a binary vector.

[0086] Table 1 Table 2 Table 3 Table 4 2. Analysis of Agrobacterium-mediated tomato transformation and guided editing efficiency. Agrobacterium-mediated transformation of tomatoes was performed according to the instructions of Vu et al. This invention uses Agrobacterium tumefaciens strain Ver. 2 (Nonaka et al., 2019, Front Plant Sci. 10: 1204) based on GV3101::pMP90, EHA105, and EHA105. Seven-day-old cotyledons of tomato plants were cut and used in the transformation. Agrobacterium containing the PE plasmid was cultured, harvested by centrifugation, and then resuspended in ABM-MS solution supplemented with 100 μM acetylsyleugenol (Vu et al., 2020) to allow OD to be adjusted. 600nm The value was 0.8. Before transformation, Agrobacterium was activated by culturing at 28°C for 1 hour. After mixing Agrobacterium with cotyledons and culturing at room temperature for 20 minutes, the transformed plants were co-cultured for 2 days before washing and then transferred to selection medium. Samples were cultured at 31°C for 5 days, then at 28°C for 5 days, and then at 25°C for the remaining steps. Newly differentiated plants were screened in a medium containing 80 mg / L kanamycin and induced to root before transplanting into soil. Hardened plants were then used for PE efficiency evaluation. For temperature treatment experiments, after co-culturing, explants were cultured at other temperatures for 5 days, then at 28°C for 5 days, and then transplanted at 25°C for the remaining steps.

[0087] To assess PE efficiency, samples were collected and targeted deep sequencing was performed on day 16 post-transformation (dpt). Leaves of the transformants were collected at all plant growth stages, and precise PE alleles were screened using PCR and Sanger sequencing.

[0088] 3. Targeted deep sequencing Genomic DNA (gDNA) was extracted from cotyledons or leaves of plants using the CTAB method. The analysis was performed using the MiniSeq sequencing service (MiniSeq™ System, Illumina, USA). MiniSeq samples were prepared using three PCRs with primers listed in Tables 5 and 6. The third PCR was performed using primers provided by the manufacturer, and sample IDs were assigned. Subsequently, the MiniSeq raw data FASTQ files were analyzed using the parameters listed in Tables 7 and 8 via the Cas-Analyzer (Park et al., 2016, Bioinformatics 33: 286-288) and CRISPResso2 (Clement et al., 2019, Nat Biotechnol. 37: 224-226).

[0089] Table 5 Table 6 Table 7 Table 8 4. Identify transformants containing the PE allele. PE allele screening was performed on transformants that survived the hardening-off stage. Three different leaf fragments from each plant were collected and combined, and gDNA was isolated using the CTAB method. PCR was used to amplify adjacent DNA sequences at the target sites, and the PCR products were sequenced using Sanger sequencing. Sequencing chromatograms were analyzed using the ICESynthgo tool to screen for potential events involving the transport of PE alleles. Targeted deep sequencing will be used to examine a subset of representative PE events in the future.

[0090] 5. Assess the presence or absence of T-DNA and replicons within the PE event. To assess the presence or absence of T-DNA and replicons, gDNA from PE plants was used as a template for PCR reactions using primer pairs explicitly indicated at the right boundary (RB) of the T-DNA and the circularized morphology of the replicons. The replicons primers were designed to amplify only circularized DNA formed in a manner released from the vector (Vu et al., 2020). PCR products were developed on 1% agarose gels, and the presence or absence of T-DNA and replicons was determined by confirming PCR strips of sizes corresponding to the T-DNA and replicons, respectively.

[0091] 6. Off-target analysis To retrieve tomato genome databases containing fewer than four segments inconsistent with the gRNA sequence. Solanum lycopersicum Potential off-target sites within (SL2.4) were identified using the Cas-OFinder tool for the gRNA sequence of the pegRNA. Cotyledonary explants 16 days post-transformation (16-dpt) with confirmed pegRNAs possessing potential off-target sites were analyzed by targeted deep sequencing using primers listed in Tables 5 and 6.

[0092] 7. Data Analysis All experiments were conducted with a minimum of three times the number of participants. Results, statistical analyses, and scatter plots were processed using MS Excel and GraphPad Prism 9.0 software. Multiple comparisons were performed using the uncorrected Fisher's LSD test.

[0093] Example 1. Analysis of the correction efficiency of the combination of guide editor constituent proteins and the types of pegRNA transcription regulatory promoters. The inventors constructed a tomato-based... ALS1 Gene-targeted guide editors using recombinant vectors ( Figure 2 and Figure 3 Afterwards, they were transformed into the cotyledons of the Tomato Red Glow variety using Agrobacterium-mediated transformation. Then, NGS analysis of the targeted sites was used to analyze whether base correction was performed.

[0094] As a result, Figure 4 As shown, it can be confirmed that the correction efficiency in the experimental group using vectors that regulate pegRNA expression via the U6-26 core promoter is significantly better than that in the experimental group using vectors that regulate pegRNA transcription via the U6 complex promoter. It can be seen that the expected base corrections occurred most frequently in the experimental group using bispecific PE2max, PE2max + Supp.gRNA and ePEmax2 guide editors.

[0095] From tomatoes ALS1 After redifferentiating into plants from gene-corrected callus, the presence or absence of the expected guided editing was reconfirmed in the redifferentiated plants. The results showed similar efficiency to the guided editing confirmed in callus, with excellent frequency and efficiency confirmed in corrected plants using guided editors PE2max, PE2max+Supp.gRNA, and ePEmax2. Figure 5 Furthermore, PCR analysis of extracted genomic DNA from redifferentiated plants to confirm the presence or absence of T-DNA and replicons indicates that T-DNA and replicons were not detected in some PE0 event plants. Figure 6 ).

[0096] Example 2. Analysis of the guided editing efficiency of transformation-mediated strains. To analyze the guided editing efficiency of Agrobacterium strains, the inventors constructed recombinant vectors using PE2max, PE2max+Supp.gRNA, or PE4max. Figure 7After transforming them into Agrobacterium superagro ver.2 (EHA105sv2), GV3101::pMP90 or EHA105 strains respectively, the guided editing efficiency was analyzed.

[0097] The results of NGS analysis in the callus stage were compared to demonstrate the efficiency of guided editing. Figure 8 As shown, the correction efficiency using strains EHA105sv2 and EHA105 was 1.27–1.43 times and 1.2–1.55 times better, respectively, than that using strain GV3101::pMP90. Comparing the frequency and efficiency of guided editing in redifferentiated plants, the results confirmed that strains EHA105sv2 and EHA105 were 0.92–4.33 times and 1.34–3.17 times better, respectively, than those using strain GV3101::pMP90. Figure 9 ).

[0098] Example 3. Analysis of the efficiency of guided editing with the delivery method of the guided editor tool. The inventors compared the delivery methods of the bootstrap editor tools, specifically comparing the bootstrap editing efficiency with T-DNA-based or geminivirus-based replicons. After cloning the same structural elements into T-DNA-based or replicons-based recombinant vectors respectively ( Figure 10 They were transformed into tomato plants using Agrobacterium EHA105sv2 strain, and then the guide editing efficiency was analyzed.

[0099] The efficiency of guided editing was compared through NGS analysis at the callus stage, such as... Figure 11 As shown, compared with the conditions using T-DNA vectors, the guided editing efficiency using twin-virus-based replication subsystems is 6.55 to 7.79 times higher.

[0100] Example 4. Analysis of guided editing efficiency under temperature conditions. In this embodiment, the guided editing efficiency of plant tissues was evaluated within 5 days after co-culturing with Agrobacterium with varying treatment temperatures (25°C, 28°C, 31°C, and 34°C). In simple terms, recombinant vectors based on replicons containing PE2max or ePEmax2 guided editors were used. Figure 12 After being transformed into tomato plants using Agrobacterium EHA105sv2, the efficiency of guided editing was analyzed.

[0101] The analysis results confirm that the efficiency of guided editing during the callus stage is directly proportional to the increase in processing temperature. Figure 13Furthermore, analysis of the frequency and efficiency of guided editing in redifferentiated plants confirmed that PE2max-guided editing plants exhibited the highest frequency of guided editing at 31°C, while ePEmax2-guided editing plants showed the highest frequencies at both 31°C and 34°C. Figure 14 Therefore, considering the efficiency of guided editing and the redifferentiation of the plant, a temperature of 31℃ is appropriate.

[0102] Example 5. Comparison of guided editing efficiency for multiple target genes The inventors compared the guided editing efficiency among target genes by performing guided editing on multiple target gene locations in tomatoes. A schematic diagram of the replicon-based vector used in this embodiment is shown below. Figure 15 As shown, the efficiency of guided editing was analyzed after Agrobacterium EHA105sv2 strain was transformed into tomato plants.

[0103] Analysis results, such as Figure 16 As shown, differences in guided editing efficiency can be confirmed among different target genes. Of the 11 genes, 7 target genes showed editing efficiencies of over 2%, while 4 genes showed editing efficiencies of less than 1%. DMR6 The gene did not induce guided editing. These results indicate that guided editing can be applied to various locations and positions within the tomato plant.

[0104] Example 6. Comparing the efficiency of terminating sub-types of the guide editor expression box To analyze the differences in guide editing efficiency with variations in the types of terminators contained within the expression cassette of the guide editor protein, a design was developed as follows: Figure 17 The guide editor protein expression box shown is constructed as follows: Figure 18 The recombinant vector based on the replicator was transformed into tomato plants using Agrobacterium EHA105sv2 strain, and the guide editing efficiency was analyzed.

[0105] The analysis results showed that t35S exhibited the lowest efficiency, but no significant difference was identified with respect to the type of terminator. Figure 19 ).

[0106] Example 7. Comparing the efficiency of different types of starters for the bootloader expression box. In this experiment, to analyze the differences in guided editing efficiency with the types of promoters that regulate the transcription of guided editor proteins, the following design was employed: Figure 20 The guide editor protein expression box shown is constructed as follows: Figure 21 The recombinant vector based on the replicator was transformed into tomato plants using Agrobacterium EHA105sv2 strain, and the guide editing efficiency was analyzed.

[0107] The analysis results showed that pSlEF1 exhibited the lowest efficiency, but no significant differences were identified with respect to promoter type. Figure 22 ).

[0108] Example 8. Comparison of editing efficiency guided by RNA chaperones In this experiment, the differences in guide editing efficiency were analyzed based on the presence, type, or arrangement order of RNA chaperone proteins within the guide editor structure. Using PE2max, which demonstrated excellent guide editing efficiency as confirmed in Example 1, as the basic structure, recombinant vectors were constructed by adding a nucleocapsid (NC) or L1ORF1p to the guide editor, or by changing the arrangement order of these proteins within the guide editor. Figure 23 and Figure 24 ).

[0109] After transforming the prepared recombinant vector into tomato plants using Agrobacterium EHA105sv2 strain, the results of guided editing efficiency were analyzed. The experimental group using the PE2max-NC guided editor showed the highest overall editing efficiency. Figure 25 Furthermore, the experimental group using the PE2max-NC bootloader editor also confirmed that the expected bootloader editing efficiency was the highest. Figure 26 ).

[0110] The results above show that using the guide editor that integrates nCas9max-MMLV RT-NC can significantly improve the efficiency of guide editing in plants.

[0111] Example 9. Analysis of correction efficiency for changes in protein composition as the guide editor changes. In this experiment, a novel guide editor expression cassette was prepared by changing the Cas9 nicking enzyme and / or MMLV reverse transcriptase used in the guide editor of PE2max or PE2max-NC. Figure 27 ), construct a target containing the above expression boxes SlCAB13 After gene recombination vector ( Figure 28 The prepared recombinant vector was transformed into tomato plants using Agrobacterium EHA105sv2 strain, and the guided editing efficiency was analyzed. The results confirmed that the editing efficiency was higher in the experimental groups using the PE6c and PE6c-NC guided editors compared to the experimental group using the PE2max guided editor. Figure 29 Furthermore, it was observed that editing efficiency was increased in the experimental group using the PE6d-NC boot editor compared to the experimental group using the PE6d boot editor.

[0112] Comparing the editing efficiency results of the PE2max-NC boot editor (which demonstrates higher editing efficiency than the PE2max boot editor in Example 8) with that of the PE6c boot editor, as shown in Example 8, the results are as follows: Figure 30 As shown, higher editing efficiency was confirmed in the experimental group using the PE6c boot editor, especially the expected boot editing efficiency.

[0113] To test the improved boot editing efficiency of the PE6c boot editor, the inventors prepared a method to remove... SlCAB13 Recombinant vectors targeting tomato genes other than the tomato gene ( Figure 31 The efficiency of guided editing was analyzed, using the PE2max guided editor as a comparison control. The analysis results are as follows: Figure 32 As shown, for all six genes tested, the PE6c bootloader editor demonstrated higher boot editing efficiency than the PE2max bootloader editor. SlHKT1;2 The gene was confirmed to be up to 8.9 times higher.

[0114] by SlHKT1;2 Gene-targeted construction of expression boxes containing multiple PE6 bootstrap editors (reference) Figure 27 Recombinant vectors ( Figure 33 The analysis of boot editing efficiency results confirmed that the PE6c-NC and PE6ec-NC boot editors showed approximately 1.5% higher boot editing efficiency than the PE6c boot editor. Figure 34 ).

[0115] The inventors have constructed an expression box comprising PE6c, PE6c-NC, and PE6ec-NC guided editors. SlOR , SlCAB13 and SlCENH3 ( sub Gene-targeted recombinant vectors ( Figure 35 After analyzing the boot editing efficiency, the three PE6 boot editors showed higher boot editing efficiency than the PE2max boot editor, except... SlCENH3 ( sub Aside from genes, the PE6c boot editor exhibits the highest efficiency, confirming that the PE6c boot editor is effective against... SlCAB13 The guided gene editing efficiency reached 14.68% ( Figure 36 ).

[0116] Example 10. Analysis of the correction efficiency of a guide editor using Arabidopsis thaliana as an example. This experiment used Arabidopsis thaliana ( Arabidopsis thaliana Using plant matter as the object, the editing efficiency of the PE2max-NC, PE6c, or PE6c-NC guided editors was analyzed. AtPDS3For the target gene, the recombinant vector constructed as described in the embodiments was transformed into plants using Agrobacterium EHA105sv2 strain.

[0117] Table 9 Analysis results, for AtPDS3 The efficiency of gene-guided editing was confirmed to be higher in both PE2max-NC and PE6c-NC guide editors compared to the PE6c guide editor, with PE2max-NC and PE6c-NC guide editors showing similar levels. Figure 37 ).

[0118] The inventors used PE2max-NC and PE6c-NC to bootstrap editors to AtCENH3 , AtOR and AtALS The results of additional gene-targeted analysis to guide editing efficiency can be achieved... AtALS and AtCENH3 The high boot editing efficiency of the PE6c-NC boot editor was confirmed in the gene. Figure 38 ).

[0119] Based on the results, it is inferred that the PE2max-NC, PE6c, PE6c-NC, PE6ec, or PE6ec-NC guide editors can be effectively used to improve guide editing efficiency in dicotyledonous plants.

Claims

1. A recombinant vector for plant-guided editing with increased correction efficiency, characterized in that, Contains virus-based replicons. The virus-based replicon includes: Guided editing RNA expression cassettes regulated via the U6 complex promoter; and Guide the editor's expression box.

2. The recombinant vector for plant-guided editing with increased correction efficiency according to claim 1, characterized in that, The viruses mentioned are: bean yellow dwarf virus, corn streak virus, tobacco mosaic virus, cauliflower mosaic virus, tobacco ringspot virus, tobacco etch virus, potato spindle tuber viroid, cucumber mosaic virus, papaya ringspot virus, tobacco streak virus, pea auricularia mosaic virus, potato virus X, potato virus Y, potato leafroll virus, cowpea mosaic virus, common bean mosaic virus, beet crescent virus, alfalfa mosaic virus, tomato spotted wilt virus, beet yellowing virus, cucumber green mottle mosaic virus, turnip yellowing mosaic virus, tobacco vein mottle virus, soybean mosaic virus, rice Donggero rod-shaped virus, rice stripe virus, rice dwarf virus, corn chlorotic mottle virus, corn dwarf mosaic virus, corn chlorotic dwarf virus, barley stripe mosaic virus, wheat streak mosaic virus, wheat dwarf virus, or wheat streak mosaic virus.

3. The recombinant vector for plant-guided editing with increased correction efficiency according to claim 1, characterized in that, The U6 complex promoter consists of the base sequence of SEQ ID NO:

10.

4. The recombinant vector for plant-guided editing with increased correction efficiency according to claim 1, characterized in that, The bootstrap editor expression box modulates the expression using the CaMV 35S promoter and the EURb7 terminator.

5. The recombinant vector for plant-guided editing with increased correction efficiency according to claim 1, characterized in that, The guide editor is: a fusion protein 1 of Cas9 cleavage enzyme protein, Moloney murine leukemia virus reverse transcriptase and nucleocapsid protein; a fusion protein 2 of Cas9 cleavage enzyme protein and Tf1 reverse transcriptase protein; or a fusion protein 3 of Cas9 cleavage enzyme protein, Tf1 reverse transcriptase protein and nucleocapsid protein.

6. The recombinant vector for plant-guided editing with increased correction efficiency according to claim 5, characterized in that, The fusion protein 1 is encoded by the base sequence of SEQ ID NO: 255, the fusion protein 2 is encoded by the base sequence of SEQ ID NO: 260 or SEQ ID NO: 264, and the fusion protein 3 is encoded by the base sequence of SEQ ID NO: 261 or SEQ ID NO:

265.

7. The recombinant vector for plant-guided editing with increased correction efficiency according to claim 1, characterized in that, The replicon also includes a filter marker expression box.

8. A method for improving the efficiency of guided editing of plant bodies, characterized in that, The step includes transforming plant cells using a recombinant vector for plant body guided editing, whose correction efficiency is increased according to any one of claims 1 to 7.

9. The method for improving the efficiency of guided editing of plant bodies according to claim 8, characterized in that, The transformation was mediated by strains with a genetic background of Agrobacterium tumefaciens EHA105 or Agrobacterium supergene ver.

2.

10. The method for improving the efficiency of guided editing of plant bodies according to claim 8, characterized in that, The transformation was mediated by Agrobacterium tumefaciens strain, and the plant tissue was treated with Agrobacterium tumefaciens at a temperature of 30-35°C after co-culturing with Agrobacterium tumefaciens.

11. A composition for improving the efficiency of guided editing correction in plants, characterized in that, The recombinant vector for plant-guided editing, which has increased correction efficiency according to any one of claims 1 to 7, is included as an active ingredient.

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