Method for increasing prime editing efficiency in plant

Abstract

The present invention relates to a method for increasing prime editing efficiency in plants. More specifically, the present invention relates to a recombinant vector for plant prime editing with enhanced editing efficiency, and a use thereof, wherein the vector comprises a virus-based replicon including: a prime editing guide RNA (pegRNA) expression cassette regulated by a U6 composite promoter; and a prime editor expression cassette.

Description

How to Increase Prime Editing Efficiency in Plants

[0001] The present invention relates to a method for increasing the efficiency of prime editing in plants.

[0002] Prime editing, a CRISPR-Cas9 tool first described by Anzalone et al. in 2019 (Nature 2019, 576, 149-157), has been reported to have the potential to repair approximately 89% of human pathogenic genetic variations, as it can accurately correct all types of point mutations, insertions, and deletions without inducing double-strand breaks (DSBs).

[0003] Prime editing technology requires two main components: the prime editor (PE) protein, which consists of a reverse transcriptase fused to the nickase Cas9 (nCas9), and the prime editing guide RNA (pegRNA), which guides PE to the target site and serves as a template for the desired correction. The pegRNA consists of a primer binding site (PBS), which serves as a docking site for the reverse transcriptase, and a reverse transcription template (RTT), which contains the desired correction information.

[0004] Since the first report of PE by Anzalone et al., improvements and applications of PE have been reported in various organisms. All of the original PE components have recently been modified and new features added, resulting in improved prime editing efficiency. However, PE application in plants has shown inconsistencies across different loci and plant species, and much work remains to be done to improve the efficiency of prime editing, particularly in dicot plants.

[0005] Accordingly, the present invention aims to provide a method for improving the efficiency of prime editing in plants, particularly dicotyledonous plants.

[0006] Meanwhile, Korean Patent Publication No. 2022-0112698 discloses a 'composition for prime editing with improved correction efficiency', and Korean Patent Publication No. 2023-0075420 discloses 'prime editing using HIV reverse transcriptase and Cas9 or a variant thereof', but there is no description of the 'method for increasing prime editing efficiency in a plant' of the present invention.

[0007] The present invention aims to provide a method for increasing the efficiency of prime editing in plants by testing various conditions, such as combinations of various prime editor component proteins, types of transcriptional regulatory factors (promoters, terminators) of the prime editor component proteins and / or pegRNA, and delivery systems, and suggesting optimized combinations.

[0008] To solve the above problem, the present invention provides a recombinant vector for plant prime editing with increased editing efficiency, comprising a virus-based replicon comprising a prime editing guide RNA (pegRNA) expression cassette controlled by a U6 composite promoter; and a prime editor expression cassette.

[0009] In addition, the present invention provides a method for improving the prime editing efficiency of a plant, comprising a step of transforming a plant cell with the recombinant vector.

[0010] In addition, the present invention provides a composition for improving the efficiency of prime editing correction in a plant, comprising the recombinant vector as an active ingredient.

[0011] Since the method according to the present invention can improve the efficiency of prime editing in plants, particularly in dicotyledonous plants, the method of the present invention can be usefully utilized in the field of new variety breeding based on precision gene editing technology.

[0012] Figure 1 is a schematic diagram of the prime editor (PE) expression cassette used in the present 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: RNaseH domain truncated mutant MMLV reverse transcriptase (D200N, T306K, W313F, T330P, L603W), MHL1dn: truncated MLH1 D754-756 lacking endonuclease domain, NC; viral nucleocapsid protein, EURb7: EU + Rb7 dual terminator, MCP-MMLV-RT: MS2 coat protein fused MMLV-RT (MCP was cloned from pREDIT_MCP-RecT, MMLV RT is a tobacco codon-optimized MMLV RT from the PE2).

[0013] FIG. 2 and FIG. 3 are schematic diagrams of recombinant vectors for prime editing used in embodiments of the present invention. FIG. 2 is a vector in which the expression of pegRNA is controlled by the U6-26 core promoter, and FIG. 3 is a vector in which the expression of pegRNA is controlled by the U6 composite promoter. Supp. gRNA refers to a gRNA that binds to a position close to pegRNA for ALS1 prime editing and assists the prime editing reaction, and 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, and pegR1.8 is composed of gRNA and the base sequence of RTT+PBS (Table 1) linked to both ends of the Cas9 gRNA scaffold sequence (corresponding to bases 92 to 177 in the base sequence of SEQ ID NO: 258).

[0014] Figure 4 shows the results of the correction efficiency analysis of prime editing using the recombinant vectors of Figures 2 and 3. *: p < 0.05, ***: p < 0.0002, ****: p < 0.0001.

[0015] Figure 5 shows the results of analyzing the frequency and efficiency of intended prime editing in regenerated plants. The y-axis numbers in the graph represent the frequency (%) of prime editing analyzed by Sanger sequencing of the target locus in transformants (plants regenerated from cotyledon explants transformed with the vectors of Figure 2 or Figure 3, i.e., PE0 events), and the numbers displayed at the top of each plot represent the efficiency of intended prime editing at the plant stage, which was calculated by dividing the number of transformants carrying intended prime editing by the total number of transformants analyzed.

[0016] Figure 6 shows the PCR results confirming the presence of replicon and T-DNA in some PE0 event plants.

[0017] Figure 7 is a schematic diagram of the recombinant vector used to compare prime editing efficiency according to Agrobacterium strains.

[0018] Figure 8 shows the results of prime editing efficiency analysis according to Agrobacterium strains. *: p < 0.05.

[0019] Figure 9 shows the results of analyzing the frequency and efficiency of intended prime editing in redifferentiated plants in a prime editing experiment according to Agrobacterium strain.

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

[0021] Figure 11 shows the results of prime editing efficiency analysis according to T-DNA or replicon-based recombinant vectors. ***: p < 0.0002, ****: p < 0.0001.

[0022] Figure 12 is a schematic diagram of a recombinant vector used to compare prime editing efficiency according to the temperature conditions under which prime editing is performed.

[0023] Figure 13 shows the results of analysis of prime editing efficiency according to the temperature conditions under which prime editing was performed. **: p < 0.0021, ***: p < 0.0002, ****: p < 0.0001.

[0024] Figure 14 shows the results of analyzing the frequency and efficiency of intended prime editing in redifferentiated plants in a prime editing experiment according to the temperature conditions under which prime editing was performed.

[0025] Figure 15 is a schematic diagram of the recombinant vector used to compare prime editing efficiency at various positions and sites. npegRNA consists of the RTT-tevopreQ1-HDV sequence of the gRNA-altered (F+E) SpCas9 gRNA scaffold-target gene of the tRNA(Gly)-target gene, and has the base sequence of SEQ ID NO: 259.

[0026] Figure 16 shows the results of prime editing efficiency analysis for various target locations and positions of a tomato plant.

[0027] Figure 17 is a schematic diagram of a prime editor expression cassette containing different terminators.

[0028] Figure 18 is a schematic diagram of a recombinant vector used to compare prime editing efficiency according to the type of terminator of the prime editor expression cassette.

[0029] Figure 19 shows the results of analysis of prime editing efficiency according to the type of terminator of the prime editor expression cassette.

[0030] Figure 20 is a schematic diagram of a prime editor expression cassette containing different promoters.

[0031] Figure 21 is a schematic diagram of a recombinant vector used to compare prime editing efficiency according to the promoter type of the prime editor expression cassette.

[0032] Figure 22 shows the results of analysis of prime editing efficiency according to the promoter type of the prime editor expression cassette.

[0033] Figure 23 is a schematic diagram of prime eddies with different presence, type, and sequence of RNA chaperone proteins.

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

[0035] Figure 25 shows the results of analysis of prime editing efficiency according to the presence, type, and sequence of RNA chaperone proteins.

[0036] Figure 26 is the result of analyzing the editing type of the target position in the results of Figure 25.

[0037] Figure 27 is a schematic diagram of a prime editor expression cassette PE6 comprising PE2max and other nickases Cas9 and / or reverse transcriptase. The coding sequences of the PE6c, PE6c-NC, PE6d, PE6d-NC, PE6ec, PE6ec-NC, PE6fc, PE6fc-NC, PE6gc, and PE6gc-NC prime editors are sequentially composed of base sequences of SEQ ID NOs: 260 to 269. 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 is a schematic diagram of a recombinant vector used to compare prime editing efficiency according to the type of PE6 prime editor expression cassette.

[0039] Figure 29 shows the results of an analysis of prime editing efficiency according to the type of PE6 prime editor expression cassette. PE2max was used as a comparative control group.

[0040] Figure 30 shows the results comparing the prime editing efficiency of the PE2max-NC prime editor and the PE6c prime editor.

[0041] Figure 31 is a schematic diagram of recombinant vectors targeting six tomato genes used to verify the enhanced editing efficiency of the PE6c prime editor.

[0042] Figure 32 shows an analysis of the prime editing efficiency of the PE6c prime editor targeting six tomato genes, with PE2max used as a comparative control.

[0043] Figure 33 is a schematic diagram of a recombinant vector containing various PE6 prime editor expression cassettes targeting the SlHKT1;2 gene.

[0044] Figure 34 shows the results of analysis of the efficiency of SlHKT1;2 gene target prime editing using various PE6 prime editors.

[0045] Figure 35 is a schematic diagram of SlOR, SlCAB13 and SlCENH3(sub) gene targeting recombination vectors containing PE6c, PE6c-NC or PE6ec-NC prime editor expression cassettes.

[0046] Figure 36 shows the analysis of the prime editing efficiency of PE6c, PE6c-NC, and PE6ec-NC prime editors for three tomato genes, with PE2max used as a comparative control.

[0047] Figure 37 is a schematic diagram of the recombinant vector used and a graph of the prime editing efficiency analysis results of PE2max-NC, PE6c or PE6c-NC prime editor targeting the AtPDS3 gene in Arabidopsis thaliana plants.

[0048] Figure 38 shows the results of an analysis of the editing efficiency of PE2max-NC or PE6c-NC prime editor targeting AtCENH3, AtOR, and AtALS genes in Arabidopsis plants, including a schematic diagram of the recombinant vector used and a prime editing efficiency analysis table.

[0049] In order to achieve the object of the present invention, the present invention provides a recombinant vector for plant prime editing with increased editing efficiency, comprising a virus-based replicon comprising a prime editing guide RNA (pegRNA) expression cassette controlled by a U6 composite promoter; and a prime editor expression cassette.

[0050] A prime editor is a type of CRISPR / Cas9-based gene-editing system that can introduce genetic changes by cutting only a single strand of DNA without double-stranded DNA cleavage. The prime editor may comprise a Cas nickase-reverse transcriptase (RT) fusion protein and a prime editing guide RNA (pegRNA), and additional domains or proteins may be additionally included in the fusion protein to enhance the efficiency of the prime editing.

[0051] The term "pegRNA" as used herein includes a guide sequence (or spacer sequence) that recognizes a target sequence, a trans-activating crispr RNA (tracrRNA) scaffold sequence, a primer binding site (PBS) required for initiating reverse transcription, and a reverse transcription template (RTT) containing a desired genetic change. In addition, the pegRNA may also include a modified form, such as a tevopreQ1 fusion, which can enhance the stability of the pegRNA.

[0052] In the above pegRNA, the guide sequence refers to a sequence within the guide RNA that designates a target site and includes a sequence that is fully or partially complementary to the target sequence. The guide sequence is any polynucleotide sequence that hybridizes with the target DNA sequence and has sufficient complementarity with the target polynucleotide sequence to induce sequence-specific binding of the gene editing complex to the target DNA sequence.

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

[0054] The term "replicon" in the present invention refers to a replication unit that controls itself. A replication unit is a continuous DNA molecule, and replication begins at a specific site within the molecule and proceeds sequentially until it is completed. Plasmids, viral DNA, and bacterial chromosomes are all replication units.

[0055] In the recombinant vector according to the present invention, the virus-based replicon may include, but is not limited to, 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), Potato leafroll virus (PLRV), Cowpea mosaic virus (CPMV), Bean common mosaic virus (BCMV), Beet curly top virus (BCTV), Alfalfa mosaic virus (AMV), Tomato spotted wilt virus (TSWV), Beet yellows virus (BYV), Cucumber green mottle mosaic virus (CGMMV), Turnip yellow mosaic virus (TYMV), Tobacco vein mottling virus (TVMV), Soybean mosaic virus (SMV), Rice tungro bacilliform virus (RTBV), Rice stripe virus (RSV), Rice dwarf virus (RDV), Maize streak virus (MSV), Maize chlorotic mottle virus (MCMV), Maize dwarf mosaic virus (MDMV), Maize chlorotic dwarf virus (MCDV),It may be a replicon based on Barley stripe mosaic virus (BSMV), Wheat streak mosaic virus (WSMV), Wheat dwarf virus (WDV) or Wheat streak mosaic virus (WSMV), and preferably a replicon based on a geminivirus such as BeYDV or MSV, but is not limited thereto.

[0056] The above replicon may include, but is not limited to, 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) into which a foreign gene to be expressed can be inserted; an SIR; and LIRs sequentially linked. The LIR of a virus has functions similar to a replication origin and a promoter, and the SIR has functions similar to a terminator. The virus-based replicon vector may be referred to the inventor's previous results (Korean Patent No. 2074744).

[0057] In the present invention, a virus-based replicon is loaded onto T-DNA, and after being injected into a plant via Agrobacterium, a circular replicon is created through rolling circle replication, and the loaded recombinant sequence is expressed.

[0058] In the recombinant vector according to the present invention, the replicon may additionally include a Prime Editor expression cassette and a selection marker expression cassette.

[0059] In a recombinant vector according to one embodiment of the present invention, the prime editor may be a fusion protein 1 of a nickase Cas9 (CRISPR associated protein 9) protein, MMLV (Moloney murine leukemia virus) reverse transcriptase, and nucleocapsid protein; a fusion protein 2 of a nickase Cas9 protein and a Tf1 Retrotransposon reverse transcriptase protein; or a fusion protein 3 of a nickase Cas9 protein, a Tf1 Retrotransposon reverse transcriptase, and a nucleocapsid protein; wherein the fusion protein 1 may be encoded by a base sequence of SEQ ID NO: 255, the fusion protein 2 may be encoded by a base sequence of SEQ ID NO: 260 or SEQ ID NO: 264, and the fusion protein 3 may be encoded by a 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 prime editor encoded by the base sequence of SEQ ID NO: 255 is PE2max-NC, the prime editor encoded by the base sequence of SEQ ID NO: 260 is PE6c, the prime editor encoded by the base sequence of SEQ ID NO: 264 is PE6ec, the prime editor encoded by the base sequence of SEQ ID NO: 261 is PE6c-NC, and the prime editor encoded by the base sequence of SEQ ID NO: 265 is PE6ec-NC (see FIGS. 23 and 27).

[0061] In addition, in one embodiment of the present invention, the nickase Cas9 protein of the PE2max-NC, PE6c and PE6c-NC prime editors may be SpCas9 (Streptococcus pyogenesCas9) having R221K, N394K and D840A mutations, and the nickase Cas9 protein of the PE6ec and PE6ec-NC prime editors may be SpCas9 having K775R, H840A and K918A mutations, but is not limited thereto, and a nickase Cas9 protein (SpG-nCas9max, encoded by the base sequence of SEQ ID NO: 278) having R221K, N394K, H840A, D1135L, S1136W, G1218K, E1219Q, R1335Q and T1337R mutations may also be used.

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

[0063] The prime editor expression cassette according to one embodiment of the present invention may be one whose expression is controlled by the CaMV 35S promoter and the EURb7 terminator (EU + Rb7 dual terminator), but is not limited thereto.

[0064] The prime editor according to the present invention is characterized by having superior efficiency in prime editing in plants compared to a prime editor formed by a combination of other elements.

[0065] In addition, in the recombinant vector according to the present invention, the selection marker is used to select cells transformed by introducing the vector, and markers that confer selectable phenotypes such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or expression of surface proteins can be used. In an environment treated with a selective agent, only cells expressing the selection marker survive, so transformed cells can be selected.

[0066] The term "recombinant" as used herein refers to a cell that replicates a heterologous nucleic acid, expresses said nucleic acid, or expresses a protein encoded by a peptide, a heterologous peptide, or a heterologous nucleic acid. A recombinant cell may express a gene or gene fragment not found in the cell's native form, either in sense or antisense form. Furthermore, a recombinant cell may express a gene found in the cell's native form, but in a modified form that has been reintroduced into the cell by artificial means.

[0067] Additionally, the term "vector" is used to refer to a DNA fragment(s) or nucleic acid molecule that is delivered into a cell. A vector replicates DNA and can reproduce independently in a host cell. The term "vector" is often used interchangeably with "vector."

[0068] The term "expression cassette" as used herein refers to a nucleic acid sequence comprising one or more genes and sequences that regulate their expression, for example, any combination of various cis-acting transcriptional regulatory elements. The expression cassette of the present invention comprises three main elements: i) a promoter; ii) a second polynucleotide, which may be referred to as a "coding polynucleotide" or "coding sequence" (also referred to as a coding gene), which is operably linked to the promoter and whose transcription is directed by the promoter when the expression cassette is introduced into a cell; and iii) a terminator polynucleotide (also referred to as a transcription terminator) that directs the termination of transcription and is located immediately downstream of the second polynucleotide.

[0069] In the present invention, the term "promoter" refers to a region of DNA upstream from a structural gene and refers to a DNA molecule to which RNA polymerase binds to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions, developmental states, or cell differentiation. A constitutive promoter may be preferred in the present invention because transformant selection can be performed in various tissues at various stages. Therefore, a constitutive promoter does not limit the possibilities of selection.

[0070] In the present invention, the promoter is a promoter suitable for transformation, and may preferably be a CaMV 35S promoter, a U6-26 core promoter, a U6 composite promoter, an actin promoter, a ubiquitin promoter, a pEMU promoter, a MAS promoter, or a histone promoter, but is not limited thereto.

[0071] In the present invention, a conventional terminator can be used as the terminator, and examples thereof include, but are not limited to, EURb7 (EU + Rb7 double terminator), t3T (EU + 35S + Rb7 triple terminator), nopaline synthase (NOS) terminator, rice α-amylase RAmy1 A terminator, phaseolin terminator, and terminator of the octopine synthase gene of Agrobacterium tumefaciens.

[0072] The present invention also provides a method for enhancing the prime editing efficiency of a plant, comprising a step of transforming a plant cell with a recombinant vector of the present invention.

[0073] In the method for improving the prime editing efficiency of a plant according to the present invention, the recombinant vector is as described above.

[0074] Plant transformation refers to any method for transferring DNA into plants. Such transformation methods do not necessarily require regeneration and / or tissue culture. Transformation of plant species is now commonplace, encompassing both dicotyledonous and monocotyledonous plants. In principle, any transformation method can be used to introduce the hybrid DNA of the present invention into a suitable progenitor cell. Methods include 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 with plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185), particle bombardment of various plant elements (DNA or RNA-coated) (Klein TM et al., 1987, Nature 327, 70), transformation of plants by Agrobacterium tumefaciens by infiltration or mature pollen or microspores tumefaciens) mediated gene transfer, infection by (non-complete) viruses (EP 0 301 316), etc. A preferred method according to the present invention comprises Agrobacterium mediated DNA transfer.

[0075] In a method according to 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 above transformation is Agrobacterium tumefaciens strain-mediated transformation, and after co-cultivation with Agrobacterium tumefaciens, the plant tissue can be treated at a temperature of 30 to 35°C, preferably 30 to 32°C, and more preferably 31°C, but is not limited thereto.

[0077] The "plant cell" used in plant transformation may be any plant cell. A plant cell may be a cultured cell, cultured tissue, cultured organ, or a whole plant. "Plant tissue" includes differentiated or undifferentiated plant tissues, such as, but not limited to, roots, stems, leaves, pollen, seeds, and cancer tissues, as well as various types of cells used in culture, such as single cells, protoplasts, shoots, and callus tissues. The plant tissue may be in planta, organ culture, tissue culture, or cell culture.

[0078] In the method of the present invention, the transformed plant cells must be redifferentiated into whole plants. Any method known in the art can be used to redifferentiate transformed plants from the transformed plant cells. Techniques for redifferentiating mature plants from callus or protoplast cultures are well known in the art for numerous different species.

[0079] The present invention also provides a composition for improving the efficiency of prime editing correction in a plant, comprising the recombinant vector of the present invention as an active ingredient.

[0080] In the composition according to the present invention, the recombinant vector is as described above. The composition of the present invention is characterized by enhancing the efficiency of prime editing at a target site in a plant.

[0081]

[0082] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0083]

[0084] Experimental method

[0085] 1. Combination of PE protein elements and plasmid construction

[0086] For the production of PE proteins used in the present invention, PE2max, PE4max, nCas9 (H840A) of ePEmax1, nCas9 of ePEmax2, ePEmax3, and eRT were cloned from the 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 sequences used in ePEmax1, ePEmax2, ePEmax3, and PE2max-NC were cloned from the pH-ePPE plasmid.

[0087] For 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 evaluating the influence of promoters, several promoters (p35S, dual CaMV 35S promoter (p2x35S) (Addgene #50269) and tomato EF1α (PSLEF1α) (Niu et al., Plant Biotechnol J. 2023, 21:5-7) were used together with the EU + 35S + Rb7 triple terminator (t3T). To evaluate the influence of terminators on PE efficiency, p35S was combined with several terminators (t35S, tNOS, EURB7, t3T).

[0088] Modified epegRNAs were designed by introducing basic modifications to the epegRNA scaffold in a modified SpCas9 scaffold (Nelson et al., Nat Biotechnol. 2022, 40:402-410). Transcription of pegRNA was driven by the U6-26 core promoter (pU6-26 core) or the U6 composite promoter (Jiang et al., Genome Biol. 2020, 21:257). The edited sites / loci, gRNA, and RTT information are detailed in Table 1. pegRNAs were amplified by PCR, cloned into expression cassettes, and then ligated into binary vectors by Golden Gate assembly.

[0089] For 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), and for geminivirus replication systems, the expression cassettes were cloned into the pLSL.R.Ly vector as previously reported (Vu et al., Plant Biotechnol J. 2020, 18:2133-2143). All biocomponents [pNOS::NptⅡ::tOCS (from pICSL11024, Addgene Plasmid #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 double terminator (EURb7; SEQ ID NO: 10), EU + 35S + Rb7 triple terminator (t3T; SEQ ID NO: 11)] were obtained from Moclo (Weber et al., 2011, PLoS One. 6(2):e16765) was domesticated with a level 0 plasmid and assembled into a PE protein assembly and binary vector.

[0090]

[0091]

[0092]

[0093]

[0094]

[0095] 2. Analysis of Agrobacterium-mediated tomato transformation and prime editing efficiency.

[0096] Agrobacterium-mediated transformation of tomato was performed as described by Vu et al. In the present invention, Agrobacterium tumefaciens strains GV3101::pMP90, EHA105, and EHA105-based SuperAgro version 2 (Nonaka et al., 2019, Front Plant Sci. 10:1204) were used. Seven-day-old cotyledons of tomato plants were excised and used for transformation. Agrobacterium containing PE plasmid were cultured, harvested by centrifugation, and resuspended in ABM-MS solution (Vu et al., 2020) supplemented with 100 μM acetosyringone, and OD 600nm This was prepared to be 0.8. Agrobacteria were activated by incubating at 28°C for 1 hour before transformation. Agrobacteria and cotyledons were mixed and incubated at room temperature for 20 minutes, and the transformed plants were cocultured for 2 days before being washed and transferred onto selection media. Samples were incubated at 31°C for 5 days, then at 28°C for 5 days, and then 25°C incubation conditions were applied for the remaining stages. Redifferentiated shoots were selected on media containing 80 mg / L kanamycin and rooting was induced before transferring to soil. Hardened plants were then used to evaluate PE efficiency. For temperature treatment experiments, after coculture, explants were incubated at different temperatures for 5 days, then at 28°C for 5 days, and then transferred to 25°C in the later stages.

[0097] To evaluate PE efficiency, samples were collected 16 days post-transformation (dpt) and subjected to targeted deep sequencing. Leaves of transformants were collected at all plant growth stages and screened for PE alleles using PCR and Sanger sequencing.

[0098]

[0099] 3. Targeted deep sequencing

[0100] Genomic DNA (gDNA) was extracted from cotyledons or plant leaves using the CTAB method. Analysis was performed using the MiniSeq sequencing service (MiniSeq™ System, Illumina, USA). MiniSeq samples were prepared through three PCRs using the primers listed in Tables 5 and 6. The third PCR was performed using the primers provided by the manufacturer to assign sample IDs. Subsequently, the MiniSeq raw data FASTQ files were analyzed using Cas-Analyzer (Park et al., 2016, Bioinformatics 33:286-288) and CRISPResso2 (Clement et al., 2019, Nat Biotechnol. 37:224-226) tools with the parameters listed in Tables 7 and 8.

[0101]

[0102]

[0103]

[0104]

[0105]

[0106] 4. Identification of transformants containing the PE allele

[0107] After the hardening step, PE allele screening was performed on surviving transformants. Three different leaf fragments from each plant were collected and combined, and gDNA was isolated using the CTAB method. PCR reactions were used to amplify DNA sequences flanking the target region, and the PCR products were sequenced by Sanger sequencing. The resulting sequencing chromatograms were analyzed by the ICE Synthgo tool to screen for potential events carrying the PE allele. Some representative PE events were subsequently verified by targeted deep sequencing.

[0108]

[0109] 5. Assessing the presence of T-DNA and replicon within PE events

[0110] To assess the presence of T-DNA and replicon, gDNA from PE plants was used as a template for PCR reactions using primer pairs designated for the right border (RB) of the T-DNA and the circularized form of the replicon. The replicon primers were designed to amplify only the circularized DNA configured to be released from the vector (Vu et al., 2020). PCR products were developed on a 1% agarose gel, and the presence of T-DNA and replicon was determined by identifying PCR bands of the sizes corresponding to the T-DNA and replicon, respectively.

[0111]

[0112] 6. Off-target analysis

[0113] The gRNA sequence of the pegRNA was searched for potential off-target sites within the tomato genome database, Solanum lycopersicum (SL2.4), using the Cas-OFinder tool, with fewer than four mismatches to the gRNA sequence. Cotyledon explants transformed with pegRNA at 16 dpt (days posttransformation) with identified potential off-target sites were analyzed by targeted deep sequencing using the primers listed in Tables 5 and 6.

[0114]

[0115] 7. Data Analysis

[0116] All experiments were performed at least three times. 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.

[0117]

[0118] Example 1. Analysis of proofreading efficiency according to the type of prime editor protein combination and pegRNA transcription control promoter.

[0119] The present inventors constructed various recombinant vectors (Figs. 2 and 3) targeting the tomato ALS1 gene and transformed cotyledons of the tomato Honggwang cultivar with each of these vectors via Agrobacterium. Subsequently, the target region was analyzed for base correction using next-generation sequencing (NGS) analysis.

[0120] As a result, as shown in Fig. 4, it was confirmed that the correction efficiency was significantly superior in the experimental group in which prime editing was performed with a vector in which pegRNA transcription was controlled by a U6 composite promoter rather than a vector in which pegRNA expression was controlled by a U6-26 core promoter. Among these, it was found that the intended base correction occurred the most in the experimental group using the prime editors PE2max, PE2Max + Supp.gRNA, and ePEmax2.

[0121] After regenerating plants from calli in which the tomato ALS1 gene was corrected, we reconfirmed whether the intended prime editing occurred in the regenerated plants. As a result, we confirmed excellent frequency and efficiency in the corrected plants using the prime editors PE2max, PE2Max + Supp.gRNA, and ePEmax2, similar to the efficiency of prime editing confirmed in calli (Fig. 5). In addition, we extracted genomic DNA from the regenerated plants and performed PCR analysis to confirm the presence of T-DNA and replicon, which was confirmed to be absent in some PE0 event plants (Fig. 6).

[0122]

[0123] Example 2. Analysis of prime editing efficiency according to the transformation vector strain.

[0124] In order to analyze the prime editing efficiency according to the Agrobacterium strain, the inventors constructed a recombinant vector using PE2max, PE2max + Supp.gRNA, or PE4max (Fig. 7), and then transformed each of these into Agrobacterium EHA105 superagro ver.2 (EHA105sv2), GV3101::pMP90, or EHA105 strains, and analyzed the prime editing efficiency.

[0125] As a result of comparing the efficiency of prime editing through NGS analysis at the callus stage, as disclosed in Fig. 8, it was confirmed that the correction efficiency when using the EHA105sv2 and EHA105 strains was 1.27 to 1.43 times and 1.2 to 1.55 times better than when using the GV3101::pMP90 strain, respectively. As a result of comparing the frequency and efficiency of prime editing in regenerated plants, it was confirmed that when using the EHA105sv2 and EHA105 strains, it was 0.92 to 4.33 times and 1.34 to 3.17 times better than when using the GV3101::pMP90 strain, respectively (Fig. 9).

[0126]

[0127] Example 3. Analysis of Prime Editing Efficiency According to the Delivery Method of the Prime Editor Tool

[0128] The present inventors compared the prime editing efficiency of the Prime Editor tool depending on the delivery method, i.e., T-DNA or geminivirus-based replicon. The same components were cloned into T-DNA or replicon-based recombinant vectors, respectively (Fig. 10), and then transformed into tomato plants using the Agrobacterium EHA105sv2 strain. Prime editing efficiency was then analyzed.

[0129] As a result of comparing the efficiency of prime editing through NGS analysis at the callus stage, as disclosed in Fig. 11, the case using a geminivirus-based replicon system showed a prime editing efficiency that was 6.55 to 7.79 times higher than the condition using a T-DNA vector.

[0130]

[0131] Example 4. Analysis of Prime Editing Efficiency According to Prime Editing Performance Temperature Conditions

[0132] In this example, the prime editing efficiency of plant tissues was evaluated according to treatment temperatures (25, 28, 31, and 34°C) within 5 days of co-cultivation with Agrobacterium. Briefly, tomato plants were transformed with the Agrobacterium EHA105sv2 strain using a replicon-based recombinant vector (Fig. 12) containing the PE2max or ePEmax2 prime editor, and the prime editing efficiency was analyzed.

[0133] The analysis results confirmed that the prime editing efficiency increased in proportion to the treatment temperature at the callus stage (Fig. 13). In addition, the analysis of the frequency and efficiency of prime editing in redifferentiated plants confirmed that the prime editing frequency was highest at 31℃ for PE2max prime-edited plants and at 31℃ and 34℃ for ePEmax2 prime-edited plants (Fig. 14). Therefore, it was found that the temperature condition of 31℃ was suitable considering the efficiency of prime editing and plant redifferentiation.

[0134]

[0135] Example 5. Comparison of Prime Editing Efficiency for Various Target Genes

[0136] The present inventors performed prime editing on various target gene sites in tomato and compared the prime editing efficiency between target genes. A schematic diagram of the replicon-based vector used in this example is shown in Figure 15. After transformation of tomato plants with the Agrobacterium EHA105sv2 strain, the prime editing efficiency was analyzed.

[0137] As a result of the analysis, as disclosed in Fig. 16, it was confirmed that there were differences in the prime editing efficiency in different target genes. Among a total of 11 genes, 7 target genes showed an editing efficiency of 2% or more, 4 genes showed an editing efficiency of less than 1%, and it was found that prime editing did not occur in the case of the DMR6 gene. The above results showed that prime editing could be applied to various locations and positions in tomato plants.

[0138]

[0139] Example 6. Comparison of efficiency according to terminator type of Prime Editor expression cassette.

[0140] In order to analyze the difference in prime editing efficiency according to the type of terminator included in the expression cassette of the prime editor protein, a prime editor protein expression cassette as in Fig. 17 was designed, a replicon-based recombinant vector as in Fig. 18 was constructed, and tomato plants were transformed using the Agrobacterium EHA105sv2 strain, and the prime editing efficiency was analyzed.

[0141] As a result of the analysis, t35S showed the lowest efficiency, but no significant difference was confirmed according to the type of terminator (Fig. 19).

[0142]

[0143] Example 7. Comparison of efficiency according to promoter type of Prime Editor expression cassette.

[0144] In this experiment, in order to analyze the difference in prime editing efficiency according to the type of promoter that controls transcription of the prime editor protein, a prime editor protein expression cassette as in Fig. 20 was designed, a replicon-based recombinant vector as in Fig. 21 was constructed, and tomato plants were transformed using the Agrobacterium EHA105sv2 strain, and the prime editing efficiency was analyzed.

[0145] As a result of the analysis, pSlEF1 showed the lowest efficiency, but no significant difference was confirmed depending on the type of promoter (Fig. 22).

[0146]

[0147] Example 8. Comparison of Prime Editing Efficiency According to RNA Chaperone

[0148] In this experiment, we analyzed the difference in prime editing efficiency according to the presence, type, or sequence of RNA chaperone proteins within the prime editor structure. Based on PE2max, which showed excellent prime editing efficiency in Example 1, as the basic structure, recombinant vectors were constructed by adding NC (nucleocapsid) or L1ORF1p to the prime editor or changing the sequence of these proteins within the prime editor (Figs. 23 and 24).

[0149] The manufactured recombinant vector was transformed into tomato plants using the Agrobacterium EHA105sv2 strain, and the prime editing efficiency was analyzed. As a result, the overall editing efficiency was confirmed to be the highest in the experimental group using the prime editor of PE2max-NC (Fig. 25). In addition, the efficiency of the intended prime editing was also confirmed to be the highest in the experimental group using the prime editor of PE2max-NC (Fig. 26).

[0150] These results show that the efficiency of prime editing in plants can be significantly increased when using a prime editor fused with nCas9max-MMLV RT-NC (nucleocapsid).

[0151]

[0152] Example 9. Analysis of proofreading efficiency according to changes in the Prime Editor composition protein.

[0153] In this experiment, we created a novel prime editor expression cassette by modifying the nickase Cas9 protein or / and MMLV reverse transcriptase used in the prime editor of PE2max or PE2max-NC (Fig. 27), constructed a recombinant vector targeting the SlCAB13 gene containing the expression cassette (Fig. 28), and transformed tomato plants with the prepared recombinant vector using the Agrobacterium EHA105sv2 strain, followed by analysis of the prime editing efficiency. As a result of the analysis, it was confirmed that the experimental groups using the prime editor of PE6c and PE6c-NC had a higher editing efficiency than the experimental group using the prime editor of PE2max (Fig. 29). In addition, it was observed that the editing efficiency was increased in the experimental group using the prime editor of PE6d-NC compared to the experimental group using the prime editor of PE6d.

[0154] As a result of comparing the editing efficiency of the prime editor of PE2max-NC, which showed a higher editing efficiency than the prime editor of PE2max in Example 8, and the prime editor of PE6c, it was confirmed that the experimental group using the prime editor of PE6c had a higher, specifically intended, prime editing efficiency, as disclosed in FIG. 30.

[0155] To verify the enhanced prime editing efficiency of the PE6c prime editor, the present inventors constructed recombinant vectors targeting tomato genes other than the SlCAB13 gene (Fig. 31) and analyzed the prime editing efficiency, using the PE2max prime editor as a comparative control. As a result of the analysis, as disclosed in Fig. 32, the PE6c prime editor showed a higher prime editing efficiency than the PE2max prime editor for all six genes tested, and in the case of the SlHKT1;2 gene, it was confirmed to be 8.9 times higher.

[0156] After constructing a recombinant vector containing various PE6 prime editor expression cassettes (see Fig. 27) targeting the SlHKT1;2 gene (Fig. 33), the prime editing efficiency was analyzed, and it was confirmed that the PE6c-NC and PE6ec-NC prime editors showed a prime editing efficiency that was approximately 1.5% higher than that of the PE6c prime editor (Fig. 34).

[0157] The present inventors constructed SlOR, SlCAB13, and SlCENH3(sub) gene targeting recombination vectors (Fig. 35) containing PE6c, PE6c-NC, and PE6ec-NC prime editor expression cassettes, and analyzed the prime editing efficiency. As a result, the three PE6 prime editors showed higher prime editing efficiency than the PE2max prime editor, and except for the SlCENH3(sub) gene, the PE6c prime editor showed the highest efficiency, and the prime editing efficiency of the PE6c prime editor for the SlCAB13 gene was confirmed to be 14.68% (Fig. 36).

[0158]

[0159] Example 10. Analysis of the proofreading efficiency of Prime Editor in Arabidopsis thaliana.

[0160] This experiment analyzed the editing efficiency of PE2max-NC, PE6c, or PE6c-NC prime editors in Arabidopsis thaliana plants. The target gene was AtPDS3, and the recombinant vector constructed as in the above examples was transformed into the plants using Agrobacterium EHA105sv2 strain.

[0161]

[0162] As a result of the analysis, the prime editing efficiency for the AtPDS3 gene was confirmed to be higher in PE2max-NC and PE6c-NC prime editors than in PE6c prime editor, and PE2max-NC and PE6c-NC prime editors were shown to be at similar levels (Fig. 37).

[0163] The inventors of the present invention further analyzed the prime editing efficiency targeting the AtCENH3, AtOR, and AtALS genes using PE2max-NC and PE6c-NC prime editors, and confirmed the high prime editing efficiency of the PE6c-NC prime editor in the AtALS and AtCENH3AtALS genes (Fig. 38).

[0164] Based on the above results, it was inferred that PE2max-NC, PE6c, PE6c-NC, PE6ec or PE6ec-NC prime editors could be useful for increasing prime editing efficiency in dicotyledonous plants.

Claims

1. A recombinant vector for plant prime editing with increased editing efficiency, comprising a virus-based replicon comprising a prime editing guide RNA (pegRNA) expression cassette controlled by a U6 composite promoter; and a prime editor expression cassette. 2.제1항에 있어서, 상기 바이러스는 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), Potato leafroll virus (PLRV), Cowpea mosaic virus (CPMV), Bean common mosaic virus (BCMV), Beet curly top virus (BCTV), Alfalfa mosaic virus (AMV), Tomato spotted wilt virus (TSWV), Beet yellows virus (BYV), Cucumber green mottle mosaic virus (CGMMV), Turnip yellow mosaic virus (TYMV), Tobacco vein mottling virus (TVMV), Soybean mosaic virus (SMV), Rice tungro bacilliform virus (RTBV), Rice stripe virus (RSV), Rice dwarf virus (RDV), Maize streak virus (MSV), Maize chlorotic mottle virus (MCMV), Maize dwarf mosaic virus (MDMV), Maize chlorotic dwarf virus (MCDV),A recombinant vector for plant prime editing with increased editing efficiency, characterized by being Barley stripe mosaic virus (BSMV), Wheat streak mosaic virus (WSMV), Wheat dwarf virus (WDV) or Wheat streak mosaic virus (WSMV).

3. A recombinant vector for plant prime editing with increased editing efficiency, characterized in that in paragraph 1, the U6 composite promoter consists of a base sequence of sequence number 10.

4. A recombinant vector for plant prime editing with increased editing efficiency, characterized in that in the first paragraph, the prime editor expression cassette is expressed by a CaMV 35S promoter and a EURb7 terminator.

5. A recombinant vector for plant prime editing with increased editing efficiency, characterized in that in claim 1, the prime editor is a fusion protein 1 of a nickase Cas9 (CRISPR associated protein 9) protein, MMLV (Moloney murine leukemia virus) reverse transcriptase, and nucleocapsid protein; a fusion protein 2 of a nickase Cas9 protein and Tf1 Retrotransposon reverse transcriptase protein; or a fusion protein 3 of a nickase Cas9 protein, Tf1 Retrotransposon reverse transcriptase, and nucleocapsid protein.

6. A recombinant vector for plant prime editing with increased editing efficiency, characterized in that in paragraph 5, 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. A recombinant vector for plant prime editing with increased correction efficiency, characterized in that in claim 1, the replicon additionally comprises a selection marker expression cassette.

8. A method for improving the prime editing efficiency of a plant, comprising a step of transforming a plant cell with a recombinant vector according to any one of claims 1 to 7.

9. A method for improving the efficiency of prime editing in a plant, characterized in that in paragraph 8, the transformation is mediated by a strain having a genetic background of Agrobacterium tumefaciens EHA105 or EHA105 superagro ver.

2.

10. A method for enhancing the efficiency of prime editing in a plant, characterized in that in paragraph 8, the transformation is Agrobacterium tumefaciens strain-mediated transformation, and the plant tissue is treated at a temperature of 30 to 35°C after co-culturing with Agrobacterium tumefaciens.

11. A composition for improving the efficiency of prime editing correction in a plant, comprising the recombinant vector of any one of claims 1 to 7 as an effective ingredient.

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