KR20230046577A - Recombinant vector for base transversion of plant and uses thereof - Google Patents

Abstract

A recombinant vector for base transformation of plants of the present invention includes uracil N-glycosylase, cytidine deaminase and nickase Cas9 (nCas9) coding sequences which are sequentially linked, and transformation by injecting the recombinant vector and a vector containing a guide RNA coding sequence specific to a target base sequence into plant cells induces effective C to G base transformation, thereby being expected to be useful for developing new varieties with improved agricultural traits.

Description

Recombinant vector for base transversion of plant and uses thereof {Recombinant vector for base transversion of plant and uses thereof}

The present invention relates to a recombinant vector for base conversion of plants and its use.

In modern agriculture, increasing crop productivity and acquiring stress-resistant traits are very important tasks. Recently, genetically modified crops using the CRISPR/Cas9 system that specifically corrects only the desired gene have been developed. This CRISPR/Cas9 system is widely used in the field of gene editing technology thanks to its simplicity and efficiency, and has made a breakthrough in the field. However, the CRISPR/Cas9 system has disadvantages in that it is difficult to obtain exactly the desired DNA sequence because it uses the random recovery system of organisms after DNA cutting, and it can only be used in the direction of eliminating the function of the gene. As an alternative to overcome the disadvantages of the CRISPR/Cas9 system, a base editor is being developed.

Base editing is a technology that utilizes the target specificity of the CRISPR/Cas9 system and the characteristics of deaminase, and can cause mutations in the desired sequence without double strand break (DSB) and introduction of foreign DNA. Since 2017, ABE (A to G base editor) and CBE (C to T base editor) applicable to plants have been developed, which are bases in which purine bases are replaced with other purine bases or pyrimidine bases are replaced with other pyrimidine bases. It is a base transition. However, it has been reported that when glycosylation of uracil proceeds exceptionally during the CBE process, a small amount of base conversion is induced by base excision repair. A base transversion is the substitution of a purine base with a pyrimidine base or a pyrimidine base with a purine base. Recently, a CGBE (C to G base editor) vector capable of base conversion in human cells has been developed using this phenomenon in reverse, but studies on CGBE vectors applicable to plants have not yet been reported.

Meanwhile, Korean Patent No. 2258713 discloses 'a composition for cytosine base correction and its use', and Korean Patent No. 2151065 discloses 'a composition for base correction in animal embryos and a base correction method'. There is no description of the recombinant vector for base conversion of plants of the present invention and its use.

The present invention was derived from the above needs, and in the present invention, in order to develop a recombinant vector for C to G base conversion of rice ( Oryza sativa ) genes, uracil glycolysis in PBE vector for C to T base conversion of existing rice genes PcCGBE (plant compatible C to G base editor) vector was created by removing uracil glycosylase inhibitor (UGI) and inserting uracil N-glycosylase (UNG) optimized for rice codons. produced. Each of the vectors containing the PcCGBE vector and the OsAAT , OsALS2 , OsCKX2 or OsSPL14 gene-specific guide RNA (gRNA) encoding sequences was injected into rice protoplast cells and transformed, and then the nucleotide sequence of the target sequence region of each gene As a result of the analysis, the present invention was completed by confirming that C to G base conversion was effectively induced in the target sequence of each gene.

In order to solve the above problems, the present invention is characterized in that uracil N-glycosylase (UNG), cytidine deaminase and nCas9 (nickase Cas9) coding sequences are sequentially linked A recombinant vector for plant base conversion is provided.

In addition, the present invention provides a composition for base conversion of plants comprising the recombinant vector and a vector containing a guide RNA coding sequence specific to a target base sequence as an active ingredient.

In addition, the present invention provides a method for base conversion of plants comprising the step of transforming by injecting the composition into plant cells.

The recombinant vector for base conversion of plants of the present invention is one in which the coding sequences of uracil N-glycosylase, cytidine deaminase, and nCas9 (nickase Cas9) are sequentially linked, and the recombinant vector And when a vector containing a guide RNA coding sequence specific to a target sequence is injected into plant cells and transformed, effective C to G base conversion is induced, so it can be usefully used for the development of new varieties with improved agricultural traits. There will be.

Figure 1 is a schematic diagram of the PcCGBE (plant compatible C to G base editor) vector of the present invention, uracil glycosylase inhibitor (UGI) in the PBE vector (A) for C to T base editing of the existing rice gene was removed to construct a miniPcCGBE vector (B), and a PcCGBE vector (C) was constructed by inserting uracil N-glycosylase (UNG) optimized for rice codons. Plant base conversion was induced using the PcCGBE recombinant vector and the vector (D) containing a guide RNA coding sequence specific to the target sequence.
Figure 2 shows that PBE, miniPcCGBE or PcCGBE vectors and vectors containing gRNA coding sequences targeting OsAAT , OsALS2 , OsCKX2 or OsSPL14 genes, respectively, were injected into rice protoplasts through polyethylene glycol (PEG)-mediated transformation, and each gene It is the result of analyzing the C to G base conversion efficiency and showing the average value. Control is a control in which only PEG was injected without vector. **, *** means that the base conversion efficiency of the vector-injected protoplasts increased statistically significantly compared to the control, ** means p<0.01 and *** p<0.001.
Figure 3 is a result of analyzing the C to G base conversion efficiency for OsAAT , OsALS2 , OsCKX2 or OsSPL14 genes according to the position of cytosine in the protospacer sequence in order to find out the editing window of the PcCGBE vector. am.

In order to achieve the object of the present invention, the present invention is characterized in that uracil N-glycosylase (UNG), cytidine deaminase and nCas9 (nickase Cas9) coding sequences are sequentially linked A recombinant vector for plant base conversion is provided.

In the recombinant vector for plant base transversion of the present invention, the base transversion is substituting cytosine (C) with guanine (G), but is not limited thereto.

In addition, the base conversion may preferably be to replace the 4th to 8th cytosines in the protospacer sequence with guanine, and more preferably to substitute the 5th to 7th cytosines in the protospacer sequence with guanine. It may be, and most preferably, the 6th cytosine in the protospacer sequence may be substituted with guanine, but is not limited thereto.

The protospacer sequence is a sequence complementary to crRNA (CRISPR RNA) and refers to a sequence of 20 to 50 bases present in front (5' direction) of a protospacer adjacent motif (PAM) sequence recognized by Cas9 protein.

In the recombinant vector for plant base conversion according to an embodiment of the present invention, the cytidine deaminase means an enzyme having an activity of substituting uracil for cytosine located in the strand where the PAM sequence of the sequence of the target site is present. As such, it may preferably be rat-derived rAPOBEC1 (rat apolipoprotein B mRNA editing enzyme, catalytic polypeptide-1), but is not limited thereto.

In addition, in the recombinant vector for plant base conversion according to an embodiment of the present invention, the nCas9 is an endonuclease protein that cuts one strand of DNA, and the uracil N-glycosylase converts uracil (U) to enzymes that catalyze glycosylation.

In addition, in the recombinant vector for plant base conversion according to an embodiment of the present invention, the 10AA linker and the XTEN linker are amino acid linkers introduced for efficient action of enzymes.

In the recombinant vector for plant base conversion of the present invention, one strand of DNA is cut by nCas9, cytosine is replaced by uracil by cytidine deaminase, and uracil is replaced by guanine by uracil N-glycosylase. , characterized by inducing C to G base conversion.

In the recombinant vector for plant base conversion according to an embodiment of the present invention, the plant is a monocotyledonous plant such as rice, barley, wheat, rye, corn, sugarcane, oat, onion, or Arabidopsis thaliana, potato, eggplant, tobacco, and pepper Dicotyledonous plants such as tomato, burdock, crown daisy, lettuce, bellflower, spinach, chard, sweet potato, carrot, water parsley, cabbage, cabbage, mustard, watermelon, melon, cucumber, pumpkin, gourd, strawberry, soybean, mung bean, kidney bean, and pea It may be, preferably a monocotyledonous plant, more preferably a rice plant, but is not limited thereto.

In the recombinant vector for plant base conversion according to one embodiment of the present invention, the recombinant vector includes a nuclear localization signal (NLS) in the 5' to 3' direction; uracil N-glycosylase; 10AA linker; cytidine deaminase; XTEN linker; nCas9; and a nuclear localization signal coding sequence may be sequentially linked, preferably a nuclear localization signal (NLS); uracil N-glycosylase; 10AA linker; cytidine deaminase; XTEN linker; nCas9; And the nuclear localization signal coding sequence may consist of the nucleotide sequence of SEQ ID NO: 1. The recombinant vector includes a ubiquitin promoter in which a ubiquitin promoter and a CaMV terminator are linked to 5' and 3', respectively; nuclear localization signal (NLS); uracil N-glycosylase; 10AA linker; cytidine deaminase; XTEN linker; nCas9; and nuclear localization signal coding sequences and CaMV terminators; Sequences may be sequentially linked (see FIG. 1C), and the uracil N-glycosylase coding sequence may be optimized for rice codons (SEQ ID NO: 2), but is not limited thereto.

As used herein, the term "recombinant" refers to a cell that replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a peptide, a protein encoded by a heterologous peptide or a heterologous nucleic acid. Recombinant cells can express genes or gene segments not found in the cell's native form, either in sense or antisense form. Recombinant cells can also express genes found in cells in their natural state, but the genes have been modified and reintroduced into the cell by artificial means.

The term "vector" is used to refer to DNA fragment(s), nucleic acid molecules, which are delivered into cells. Vectors replicate DNA and can reproduce independently in host cells. The term “delivery vehicle” is often used interchangeably with “vector”.

Expression vectors preferably contain one or more selectable markers. The marker is a nucleic acid sequence having a characteristic that can be selected by a conventional chemical method, and includes all genes capable of distinguishing transformed cells from non-transformed cells. Examples include herbicide resistance genes such as zeocin (bleomycin), glyphosate, or phosphinotricin, and antibiotic resistance genes such as kanamycin, G418, hygromycin, and chloramphenicol. It is not limited.

In addition, the present invention provides a composition for plant base conversion comprising, as an active ingredient, the recombinant vector for plant base conversion and a vector containing a guide RNA coding sequence specific to a target base sequence.

In the composition for base conversion of the present invention, the recombinant vector for plant base conversion is as described above.

In the composition for plant base conversion according to an embodiment of the present invention, the target base sequence may preferably be SEQ ID NO: 3 to SEQ ID NO: 6, more preferably SEQ ID NO: 5 or SEQ ID NO: 6 However, it is not limited thereto.

In addition, the present invention provides a plant base conversion method comprising the step of transforming plant cells by injecting the plant base conversion composition.

In the base conversion method according to the present invention, the transformation method by injecting the composition into plant cells is a calcium/polyethylene glycol method for protoplasts (Krens et al., 1982, Nature 296:72-74; Negrutiu et al. , 1987, Plant Mol. Biol. 8:363-373), electroporation of protoplasts (Shillito et al., 1985, Bio/Technol. 3:1099-1102), microinjection into plant elements (Crossway et al. Gen. Genet. ens ( Agrobacterium tumefaciens ) It can be appropriately selected from infection by bacteria in (incomplete) mediated gene transfer.

In addition, introducing the composition into plant cells refers to a transformation method. Transformation of plant species is now common for plant species including both dicotyledonous as well as monocotyledonous plants. In principle, any transformation method can be used to introduce the recombinant vectors according to the invention into suitable progenitor cells.

In the base conversion method according to the present invention, the "plant cell" into which the composition is injected can be any plant cell. A plant cell is a cultured cell, cultured tissue, cultured organ or whole plant. "Plant tissue" refers to differentiated or undifferentiated plant tissue, including, but not limited to, roots, stems, leaves, pollen, microspores, ovules, seeds, and various types of cells used in culture, i.e., single cells, Includes protoplast, shoot and callus tissues. Plant tissue may be in planta or may be in organ culture, tissue culture or cell culture. Preferred plant cells according to the present invention are protoplasts.

The transgenic plants of the present invention were transformed with a recombinant vector in which uracil N-glycosylase, cytidine deaminase and nCas9 coding sequences were sequentially linked and a vector containing a guide RNA coding sequence specific to the target sequence, compared to the control group. C to G base conversion efficiency may be increased.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only to illustrate the present invention, and the content of the present invention is not limited to the following examples.

Materials and Methods

1. Plant material

Experiments were conducted using Oryza sativa L. cv. The seed coat of the seed was removed, treated with 70% (v/v) ethanol for 1 minute, and then stirred in a 50% (v/v) Clorox solution for 40 minutes to disinfect the surface. The disinfected seeds were washed more than 10 times with distilled water, and then cultured in MS (Murashige and Skoog) medium containing 1% (w/v) sucrose at 28 ° C for 10 days in dark conditions and 1 day in light conditions. .

2. Production of recombinant vectors for C to G conversion that can be used in plants

A PcCGBE (plant compatible C to G base editor) vector was prepared by modifying the PBE (plant base editor) vector disclosed in Zong et al. (2017, Nat. Biotechnol. 35:438-440). Specifically, the miniPcCGBE vector was constructed by removing the uracil glycosylase inhibitor (UGI) from the PBE vector using Mlu I and Xma I restriction enzymes, and the N-terminus of miniPcCGBE was Hind III and Pml I After linearization with a restriction enzyme, E. coli uracil N-glycosylase (UNG) optimized for rice codons was attached to this position to complete the PcCGBE vector (Fig. 1A, B and C).

3. Rice protoplast isolation

After cutting the above-ground tissue of rice into small pieces within 0.5 mm with a razor, biochemically decompose the cell wall by applying a vacuum for 4 hours in an enzyme solution (1.5% Cellulase R-10, 0.75% Macerozyme R-10) three times. and the protoplasts were isolated. After counting the number of protoplasts using a hemocytometer, 5×10 ⁶ protoplast cells were used in the experiment.

4. Rice protoplast transformation

A recombinant vector for base correction (PBE, miniPcCGBE or PcCGBE) and a vector (Fig. 1D) containing each of the guide RNA coding sequences specific to four genes ( OsAAT , OsALS2 , OsCKX2 or OsSPL14 ) were mixed at a molar concentration of 1:3. After mixing, the rice protoplast cells were injected using PEG (polyethylene glycol). After each vector was injected, the cells were cultured for 60 hours under dark conditions so that base conversion could occur in the cells.

5. Base conversion result analysis

After genomic DNA was extracted from the transformed rice protoplast cells, PCR was performed three times to prepare a template for deep sequencing. In the first PCR, gene-specific primers (Table 1) were used to amplify a target gene in the genome. The second and third PCRs are processes of attaching barcodes used for deep sequencing, and the first PCR product was used as a template. After amplicon deep sequencing using Illumina's MiniSeq equipment, analysis was performed using CRISPR RGEN tool and CRISPResso2 tool.

Example 1. Development of recombinant vector for C to G base conversion

A PcCGBE vector for C to G base conversion applicable to rice plants was constructed using the PBE vector for C to T base conversion. The PBE vector is a cytosine deaminase, and rAPOBEC1 is linked to the N-terminus of nCas9 (nikase Cas9). By this cytosine deaminase, cytosine (C) is replaced by thymine (T). Exceptionally, when glycosylation of uracil proceeds, base excision repair (BER) causes guanine (not thymine) to occur. It has been reported that it may be substituted with guanine, G). In the present invention, a PcCGBE vector was constructed from a PBE vector by using this phenomenon in reverse. In the PBE vector, a uracil glycosylase inhibitor (UGI) is linked to the C-terminus to prevent uracil glycosylation. Therefore, a miniPcCGBE vector was constructed by removing the uracil glycosylase inhibitor from the PBE vector, and a PcCGBE vector was finally constructed by adding uracil N-glycosylase (UNG) to the N-terminus of the miniPcCGBE vector (FIG. 1).

Example 2. Verification of C to G base conversion efficiency

OsAAT (XM_015765674), OsALS2 (XM_015770973.1), OsCKX2 (NM_001048837.1) to confirm the C to G nucleotide conversion efficiency of the miniPcCGBE and PcCGBE vectors of the present invention Alternatively, gRNAs (Table 2) targeting the OsSPL14 (XM_026027598.1) gene, respectively, were injected into the protoplasts together with the vectors to transform them. Thereafter, amplicon deep sequencing was performed using the genomic DNA of the protoplasts from which the base conversion was induced, and the C to G base conversion efficiency of each gene was analyzed and the average value was calculated. A transformant injected with only PEG without vector was used as a control.

gRNA sequences used in the present invention Gene Sequences (protospacer with PAM ) sequence number OsAAT CAAAA C ACCTTACCTCGGTC CGG 3 OsALS2 CAGGT C CCCCGCCGCATGAT CGG 4 OsCKX2 GAGCT C AAGCTCCGCGCCGC GGG 5 OsSPL14 CTCTT C TGTCAACCCAGCCA TGG 6

* Bold C represents the 6th cytosine base in the protospacer sequence.

As a result, it was confirmed that the average efficiency of C to G base conversion of the transformants injected with the miniPcCGBE and PcCGBE vectors of the present invention was significantly higher than that of the control group. When the PcCGBE vector was injected, the efficiency was about 0.28%, and when the miniPcCGBE vector was injected, it was confirmed that the efficiency was about 0.1% (FIG. 2). Through the above results, it was found that when UNG was added (PcCGBE vector) than when only UGI was removed (miniPcCGBE vector), the BER pathway was more induced and C to G base conversion increased.

Example 3. Analysis of base conversion efficiency according to cytosine position in protospacer sequence

In order to examine the editing window of the PcCGBE vector of the present invention, base conversion efficiencies of four genes according to cytosine positions in the protospacer sequence were analyzed.

As a result, among the OsAAT , OsALS2 , OsCKX2 or OsSPL14 genes, the base conversion efficiency of the OsCKX2 or OsSPL14 gene was remarkably high. Among the cytosines in the protospacer consisting of 20 nucleotides in all of the OsAAT , OsALS2 , OsCKX2 , and OsSPL14 genes, it was confirmed that base conversion occurs the most at cytosine (C6) at position 6. OsAAT gene and OsALS2 gene did not show a clear trend because of their overall low efficiency, but OsCKX2 gene and OsSPL14 gene showed that the most C to G base conversion occurred at cytosine at position 6 (FIG. 3).

<110> Seoul National University R&DB Foundation <120> Recombinant vector for base transversion of plant and uses its <130> PN21323 <160> 42 <170> KoPatentIn 3.0 <210> 1 <211> 5640 <212> DNA <213> artificial sequence <220> <223> PcCGBE vector <400> 1 ccaaagaaga agaggaaggt tgcgaatgag cttacatggc atgatgtcct ggctgaggag 60 aagcagcagc cgtacttctt aaatacgctg caaacagtcg ccagcgagcg gcagtcgggg 120 gtcaccatct atccaccgca gaaagatgtt ttcaacgcct tcaggtttac cgagttgggc 180 gacgtcaaag ttgtaattct tggtcaagat ccctaccacg gccctggaca agcacatggt 240 cttgcttttt cagtgaggcc gggcattgca ataccgccgt cgctattgaa catgtacaag 300 gagctggaaa atactattcc aggcttcacg cgccctaatc atggatattt ggaaagctgg 360 gcgaggcaag gtgttcttct actcaacact gttctcaccg tacgtgccgg gcaagctcat 420 tctcacgcca gtttagggtg ggagacattt actgacaagg tgatatccct catcaaccag 480 caccgcgagg gtgtggtgtt cctcctgtgg ggctctcacg cgcagaagaa gggggccatc 540 atcgacaaac agcggcacca cgtgctgaag gctccacatc catctcctct ctccgcgcac 600 aggggattct tcggctgcaa ccattttgtc ctcgcaaatc aatggctgga gcagagagga 660 gaaacgccta ttgattggat gccggtgttg cccgcagaat cagaatcggg ggggagcggc 720 ggctcggggg ggagctcatc ggagaccggc cctgttgctg ttgaccccac cctgcggcgg 780 agaatcgagc cacacgagtt cgaggtgttc ttcgacccaa gggagctccg caaggagacg 840 tgcctcctgt acgagatcaa ctggggcggc aggcactcca tctggaggca caccagccaa 900 aacaccaaca agcacgtgga ggtcaacttc atcgagaagt tcaccaccga gaggtacttc 960 tgcccaaaca cccgctgctc catcacctgg ttcctgtcct ggagcccatg cggcgagtgc 1020 tccagggcca tcaccgagtt cctcagccgc tacccacacg tcaccctgtt catctacatc 1080 gccaggctct accaccacgc cgacccaagg aacaggcagg gcctccgcga cctgatctcc 1140 agcggcgtga ccatccaaat catgaccgag caggagtccg gctactgctg gaggaacttc 1200 gtcaactact ccccaagcaa cgaggcccac tggccaaggt acccacacct ctgggtgcgc 1260 ctctacgtgc tcgagctgta ctgcatcatc ctcggcctgc caccatgcct caacatcctg 1320 aggcgcaagc aaccacagct gaccttcttc accatcgccc tccaaagctg ccactaccag 1380 aggctcccac cacacatcct gtgggctacc ggcctcaagt ccggcagcga gacgccaggc 1440 acctccgaga gcgctacgcc tgaacttaag gacaagaagt actcgatcgg cctcgccatc 1500 gggacgaact cagttggctg ggccgtgatc accgacgagt acaaggtgcc ctctaagaag 1560 ttcaaggtcc tggggaacac cgaccgccat tccatcaaga agaacctcat cggcgctctc 1620 ctgttcgaca gcggggagac cgctgaggct acgaggctca agagaaccgc taggcgccgg 1680 tacacgagaa ggaagaacag gatctgctac ctccaagaga ttttctccaa cgagatggcc 1740 aaggttgacg attcattctt ccaccgcctg gaggagtctt tcctcgtgga ggaggataag 1800 aagcacgagc ggcatcccat cttcggcaac atcgtggacg aggttgccta ccacgagaag 1860 taccctacga tctaccatct gcggaagaag ctcgtggact ccaccgataa ggcggacctc 1920 agactgatct acctcgctct ggcccacatg atcaagttcc gcggccattt cctgatcgag 1980 ggggatctca acccagacaa cagcgatgtt gacaagctgt tcatccaact cgtgcagacc 2040 tacaaccaac tcttcgagga gaacccgatc aacgcctctg gcgtggacgc gaaggctatc 2100 ctgtccgcga ggctctcgaa gtccaggagg ctggagaacc tgatcgctca gctcccaggc 2160 gagaagaaga acggcctgtt cgggaacctc atcgctctca gcctggggct caccccgaac 2220 ttcaagtcga acttcgatct cgctgaggac gccaagctgc aactctccaa ggacacctac 2280 gacgatgacc tcgataacct cctggcccag atcggcgatc aatacgcgga cctgttcctc 2340 gctgccaaga acctgtcgga cgccatcctc ctgtcagata tcctccgcgt gaacaccgag 2400 atcacgaagg ctccactctc tgcctccatg atcaagcgct acgacgagca ccatcaggat 2460 ctgaccctcc tgaaggcgct ggtccgccaa cagctcccgg agaagtacaa ggagattttc 2520 ttcgatcagt cgaagaacgg ctacgctggg tacatcgacg gcggggcctc acaagaggag 2580 ttctacaagt tcatcaagcc aatcctggag aagatggacg gcacggagga gctcctggtg 2640 aagctcaaca gggaggacct cctgcggaag cagagaacct tcgataacgg cagcatcccc 2700 caccaaatcc atctcgggga gctgcacgcc atcctgagaa ggcaagagga cttctaccct 2760 ttcctcaagg ataaccggga gaagatcgag aagatcctga ccttcagaat cccatactac 2820 gtcggccctc tcgcgcgggg gaactcaaga ttcgcttgga tgacccgcaa gtctgaggag 2880 accatcacgc cgtggaactt cgaggaggtg gtggacaagg gcgctagcgc tcagtcgttc 2940 atcgagagga tgaccaactt cgacaagaac ctgcccaacg agaaggtgct ccctaagcac 3000 tcgctcctgt acgagtactt caccgtctac aacgagctca cgaaggtgaa gtacgtcacc 3060 gagggcatgc gcaagccagc gttcctgtcc ggggagcaga agaaggctat cgtggacctc 3120 ctgttcaaga ccaaccggaa ggtcacggtt aagcaactca aggaggacta cttcaagaag 3180 atcgagtgct tcgattcggt cgagatcagc ggcgttgagg accgcttcaa cgccagcctc 3240 gggacctacc acgatctcct gaagatcatc aaggataagg acttcctgga caacgaggag 3300 aacgaggata tcctggagga catcgtgctg accctcacgc tgttcgagga cagggagatg 3360 atcgaggagc gcctgaagac gtacgcccat ctcttcgatg acaaggtcat gaagcaactc 3420 aagcgccgga gatacaccgg ctgggggagg ctgtcccgca agctcatcaa cggcatccgg 3480 gacaagcagt ccgggaagac catcctcgac ttcctcaaga gcgatggctt cgccaacagg 3540 aacttcatgc aactgatcca cgatgacagc ctcaccttca aggaggatat ccaaaaggct 3600 caagtgagcg gccagggggga ctcgctgcac gagcatatcg cgaacctcgc tggctccccc 3660 gcgatcaaga agggcatcct ccagaccgtg aaggttgtgg acgagctcgt gaaggtcatg 3720 ggccggcaca agcctgagaa catcgtcatc gagatggcca gagagaacca aaccacgcag 3780 aaggggcaaa agaactctag ggagcgcatg aagcgcatcg aggagggcat caaggagctg 3840 gggtcccaaa tcctcaagga gcacccagtg gagaacaccc aactgcagaa cgagaagctc 3900 tacctgtact acctccagaa cggcagggat atgtacgtgg accaagagct ggatatcaac 3960 cgcctcagcg attacgacgt cgatcatatc gttccccagt ctttcctgaa ggatgactcc 4020 atcgacaaca aggtcctcac caggtcggac aagaaccgcg gcaagtcaga taacgttcca 4080 tctgaggagg tcgttaagaa gatgaagaac tactggaggc agctcctgaa cgccaagctg 4140 atcacgcaaa ggaagttcga caacctcacc aaggctgaga gaggcgggct ctcagagctg 4200 gacaaggccg gcttcatcaa gcggcagctg gtcgagacca gacaaatcac gaagcacgtt 4260 gcgcaaatcc tcgactctcg gatgaacacg aagtacgatg agaacgacaa gctgatcagg 4320 gaggttaagg tgatcaccct gaagtctaag ctcgtctccg acttcaggaa ggatttccag 4380 ttctacaagg ttcgcgagat caacaactac caccatgccc atgacgctta cctcaacgct 4440 gtggtcggca ccgctctgat caagaagtac ccaaagctgg agtccgagtt cgtgtacggg 4500 gactacaagg tttacgatgt gcgcaagatg atcgccaagt cggagcaaga gatcggcaag 4560 gctaccgcca agtacttctt ctactcaaac atcatgaact tcttcaagac cgagatcacg 4620 ctggccaacg gcgagatccg gaagagaccg ctcatcgaga ccaacggcga gacggggggag 4680 atcgtgtggg acaagggcag ggatttcgcg accgtccgca aggttctctc catgccccag 4740 gtgaacatcg tcaagaagac cgaggtccaa acgggcgggt tctcaaagga gtctatcctg 4800 cctaagcgga acagcgacaa gctcatcgcc agaaagaagg actgggaccc aaagaagtac 4860 ggcgggttcg acagccctac cgtggcctac tcggtcctgg ttgtggcgaa ggttgagaag 4920 ggcaagtcca agaagctcaa gagcgtgaag gagctcctgg ggatcaccat catggagagg 4980 tccagcttcg agaagaaccc aatcgacttc ctggaggcca aggggctacaa ggaggtgaag 5040 aaggacctga tcatcaagct cccgaagtac tctctcttcg agctggagaa cggcaggaag 5100 agaatgctgg cttccgctgg cgagctccag aaggggaacg agctcgcgct gccaagcaag 5160 tacgtgaact tcctctacct ggcttcccac tacgagaagc tcaagggcag cccggaggac 5220 aacgagcaaa agcagctgtt cgtcgagcag cacaagcatt acctcgacga gatcatcgag 5280 caaatctccg agttcagcaa gcgcgtgatc ctcgccgacg cgaacctgga taaggtcctc 5340 tccgcctaca acaagcaccg ggacaagccc atcagagagc aagcggagaa catcatccat 5400 ctcttcaccc tgacgaacct cggcgctcct gctgctttca agtacttcga caccacgatc 5460 gatcggaaga gatacacctc cacgaaggag gtcctggacg cgaccctcat ccaccagtcg 5520 atcaccggcc tgtacgagac gaggatcgac ctctcacaac tcggcgggga taagagaccc 5580 gcagcaacca agaaggcagg gcaagcaaag aagaagaagc caaagaagaa gcggaaggtg 5640 5640 <210> 2 <211> 684 <212> DNA <213> artificial sequence <220> <223> rice codon optimized uracil N-glycosylase <400> 2 gcgaatgagc ttacatggca tgatgtcctg gctgaggaga agcagcagcc gtacttctta 60 aatacgctgc aaacagtcgc cagcgagcgg cagtcggggg tcaccatcta tccaccgcag 120 aaagatgttt tcaacgcctt caggtttacc gagttgggcg acgtcaaagt tgtaattctt 180 ggtcaagatc cctaccacgg ccctggacaa gcacatggtc ttgctttttc agtgaggccg 240 ggcattgcaa taccgccgtc gctattgaac atgtacaagg agctggaaaa tactattcca 300 ggcttcacgc gccctaatca tggatatttg gaaagctggg cgaggcaagg tgttcttcta 360 ctcaacactg ttctcaccgt acgtgccggg caagctcatt ctcacgccag tttagggtgg 420 gagacattta ctgacaaggt gatatccctc atcaaccagc accgcgaggg tgtggtgttc 480 ctcctgtggg gctctcacgc gcagaagaag ggggccatca tcgacaaaca gcggcaccac 540 gtgctgaagg ctccacatcc atctcctctc tccgcgcaca ggggattctt cggctgcaac 600 catttgtcc tcgcaaatca atggctggag cagagaggag aaacgcctat tgattggatg 660 ccggtgttgc ccgcagaatc agaa 684 <210> 3 <211> 23 <212> DNA <213> artificial sequence <220> <223> gRNA <400> 3 caaaaccct tacctcggtc cgg 23 <210> 4 <211> 23 <212> DNA <213> artificial sequence <220> <223> gRNA <400> 4 caggtccccc gccgcatgat cgg 23 <210> 5 <211> 23 <212> DNA <213> artificial sequence <220> <223> gRNA <400> 5 gagctcaagc tccgcgccgc ggg 23 <210> 6 <211> 23 <212> DNA <213> artificial sequence <220> <223> gRNA <400> 6 ctcttctgtc aacccagcca tgg 23 <210> 7 <211> 19 <212> DNA <213> artificial sequence <220> <223> primer <400> 7 tcgtccgtct tcgctggcc 19 <210> 8 <211> 25 <212> DNA <213> artificial sequence <220> <223> primer <400> 8 gaacacggaa tctgtacact cagcc 25 <210> 9 <211> 23 <212> DNA <213> artificial sequence <220> <223> primer <400> 9 cggtcatcac caaccacctc ttc 23 <210> 10 <211> 21 <212> DNA <213> artificial sequence <220> <223> primer <400> 10 ccaccaccga catagagaat c 21 <210> 11 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 11 gctgctagtg ctggcaaaat 20 <210> 12 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 12 acgcacaata tgaggggtcg 20 <210> 13 <211> 23 <212> DNA <213> artificial sequence <220> <223> primer <400> 13 cgctgatgtg ttgtttgttg cga 23 <210> 14 <211> 23 <212> DNA <213> artificial sequence <220> <223> primer <400> 14 cctgcagagc aagctcaagc tca 23 <210> 15 <211> 51 <212> DNA <213> artificial sequence <220> <223> primer <400> 15 acactctttc cctacacgac gctcttccga tcttcgacct gatcggtgct c 51 <210> 16 <211> 54 <212> DNA <213> artificial sequence <220> <223> primer <400> 16 gtgactggag ttcagacgtg tgctcttccg atctatccac caccaatcca atcc 54 <210> 17 <211> 52 <212> DNA <213> artificial sequence <220> <223> primer <400> 17 acactctttc cctacacgac gctcttccga tctggcaacc aacctcgtgt cc 52 <210> 18 <211> 56 <212> DNA <213> artificial sequence <220> <223> primer <400> 18 gtgactggag ttcagacgtg tgctcttccg atctgacaag gtaattgtgc ttggtg 56 <210> 19 <211> 55 <212> DNA <213> artificial sequence <220> <223> primer <400> 19 acactctttc cctacacgac gctcttccga tcttttgcag aggatggatg tgctg 55 <210> 20 <211> 54 <212> DNA <213> artificial sequence <220> <223> primer <400> 20 gtgactggag ttcagacgtg tgctcttccg atctacaggt tcagccatgg gtgc 54 <210> 21 <211> 53 <212> DNA <213> artificial sequence <220> <223> primer <400> 21 acactctttc cctacacgac gctcttccga tcttcgctgg cccaaatctc cct 53 <210> 22 <211> 54 <212> DNA <213> artificial sequence <220> <223> primer <400> 22 gtgactggag ttcagacgtg tgctcttccg atctgacatg gctgcagcct ggtt 54 <210> 23 <211> 57 <212> DNA <213> artificial sequence <220> <223> primer <400> 23 aatgatacgg cgaccaccga gatctacact atagcctaca ctctttccct acacgac 57 <210> 24 <211> 57 <212> DNA <213> artificial sequence <220> <223> primer <400> 24 aatgatacgg cgaccaccga gatctacaca tagaggcaca ctctttccct acacgac 57 <210> 25 <211> 57 <212> DNA <213> artificial sequence <220> <223> primer <400> 25 aatgatacgg cgaccaccga gatctacacc ctatcctaca ctctttccct acacgac 57 <210> 26 <211> 57 <212> DNA <213> artificial sequence <220> <223> primer <400> 26 aatgatacgg cgaccaccga gatctacacg gctctgaaca ctctttccct acacgac 57 <210> 27 <211> 57 <212> DNA <213> artificial sequence <220> <223> primer <400> 27 aatgatacgg cgaccaccga gatctacaca ggcgaagaca ctctttccct acacgac 57 <210> 28 <211> 57 <212> DNA <213> artificial sequence <220> <223> primer <400> 28 aatgatacgg cgaccaccga gatctacact aatcttaaca ctctttccct acacgac 57 <210> 29 <211> 57 <212> DNA <213> artificial sequence <220> <223> primer <400> 29 aatgatacgg cgaccaccga gatctacacc aggacgtaca ctctttccct acacgac 57 <210> 30 <211> 57 <212> DNA <213> artificial sequence <220> <223> primer <400> 30 aatgatacgg cgaccaccga gatctacacg tactgacaca ctctttccct acacgac 57 <210> 31 <211> 53 <212> DNA <213> artificial sequence <220> <223> primer <400> 31 caagcagaag acggcatacg agatcgagta atgtgactgg agttcagacg tgt 53 <210> 32 <211> 53 <212> DNA <213> artificial sequence <220> <223> primer <400> 32 caagcagaag acggcatacg agattctccg gagtgactgg agttcagacg tgt 53 <210> 33 <211> 53 <212> DNA <213> artificial sequence <220> <223> primer <400> 33 caagcagaag acggcatacg agataatgag cggtgactgg agttcagacg tgt 53 <210> 34 <211> 53 <212> DNA <213> artificial sequence <220> <223> primer <400> 34 caagcagaag acggcatacg agatggaatc tcgtgactgg agttcagacg tgt 53 <210> 35 <211> 53 <212> DNA <213> artificial sequence <220> <223> primer <400> 35 caagcagaag acggcatacg agatttctga atgtgactgg agttcagacg tgt 53 <210> 36 <211> 53 <212> DNA <213> artificial sequence <220> <223> primer <400> 36 caagcagaag acggcatacg agatacgaat tcgtgactgg agttcagacg tgt 53 <210> 37 <211> 53 <212> DNA <213> artificial sequence <220> <223> primer <400> 37 caagcagaag acggcatacg agatagcttc aggtgactgg agttcagacg tgt 53 <210> 38 <211> 53 <212> DNA <213> artificial sequence <220> <223> primer <400> 38 caagcagaag acggcatacg agatgcgcat tagtgactgg agttcagacg tgt 53 <210> 39 <211> 53 <212> DNA <213> artificial sequence <220> <223> primer <400> 39 caagcagaag acggcatacg agatcatagc cggtgactgg agttcagacg tgt 53 <210> 40 <211> 53 <212> DNA <213> artificial sequence <220> <223> primer <400> 40 caagcagaag acggcatacg agatttcgcg gagtgactgg agttcagacg tgt 53 <210> 41 <211> 53 <212> DNA <213> artificial sequence <220> <223> primer <400> 41 caagcagaag acggcatacg agatgcgcga gagtgactgg agttcagacg tgt 53 <210> 42 <211> 53 <212> DNA <213> artificial sequence <220> <223> primer <400> 42 caagcagaag acggcatacg agatctatcg ctgtgactgg agttcagacg tgt 53

Claims (10)

A recombinant vector for plant base conversion, characterized in that the coding sequences of uracil N-glycosylase (UNG), cytidine deaminase, and nickase Cas9 (nCas9) are sequentially linked. The recombinant vector for plant base conversion according to claim 1, wherein the base conversion is performed by replacing cytosine (C) with guanine (G). The recombinant vector for plant base conversion according to claim 1, wherein the base conversion is performed by substituting 6th cytosine (C) in a protospacer sequence with guanine (G). The recombinant vector for plant base conversion according to claim 1, wherein the plant is rice. The method of claim 1, wherein the recombinant vector comprises a nuclear localization signal (NLS) in the 5' to 3' direction; uracil N-glycosylase; 10AA linker; cytidine deaminase; XTEN linker; nCas9; and a nuclear localization signal coding sequence sequentially linked to each other. The recombinant vector for plant base conversion according to claim 5, wherein the uracil N-glycosylase coding sequence consists of the nucleotide sequence of SEQ ID NO: 2 optimized for rice codons. The recombinant vector for plant base conversion according to claim 6, wherein the recombinant vector comprises the nucleotide sequence of SEQ ID NO: 1. A composition for base conversion of plants comprising, as an active ingredient, the recombinant vector of any one of claims 1 to 7 and a vector containing a guide RNA coding sequence specific to a target base sequence. The composition for base conversion of plants according to claim 8, wherein the target base sequence is SEQ ID NO: 5 or 6. A method for transforming plants by injecting the composition of claim 8 into plant cells to transform them.

Images