An adenine base editing tool and its method and application

Abstract

The present invention relates to an adenine base editing tool and its method and application. The adenine base editing tool includes an NG-ABE9e expression vector, and the NG-ABE9e expression vector is plasmid pCMV-NG-ABE9e, whose sequence is shown in SEQ ID NO.1. The present invention effectively narrows its editing window while maintaining high activity and high fidelity by introducing key mutation information into the adenosine deaminase coding sequence of NG-ABE8e, so as to improve the precision of ABE editing and increase its application in the field of life.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and particularly to an adenine base editing tool with high editing efficiency and narrow scope, and its methods and applications. Background Art

[0002] Once the CRISPR / Cas (clustered regularly interspaced short palindromic repeats / CRISPR associated proteins) system came out, it quickly became a popular third-generation gene editing tool. However, when the CRISPR / Cas system edits genes, it is not very precise, so unwanted editing by-products will be generated. In view of this, the base editing technology developed based on the CRISPR / Cas system came into being. Different from the CRISPR / Cas system, the base editing technology does not rely on the generation of DNA double-strand breaks (DSBs) to play its role, and can achieve efficient and precise single-base editing without the participation of donor DNA, so the fidelity and safety are higher. For these characteristics, the base editing technology is also considered to be a safer fourth-generation gene editing tool.

[0003] At present, the base editing technology can be divided into two categories: DNA base editors and RNA base editors. In 2016, the David Liu team took the lead in developing the first form of DNA base editor - cytosine base editor (CBE), realizing the conversion of cytosine (C) to thymine (T). In 2017, the team innovatively developed the second form of DNA base editor - adenine base editor (ABE), which can achieve the conversion of adenine (A) to guanine (G). Among them, ABE is mainly composed of three elements: sgRNA, modified Cas9 protein, and artificially evolved adenine deaminase (TadA). Similar to the working principle of CBE, the ABE system first specifically pairs with the complementary strand of the target sequence under the guidance of sgRNA, and then through the action of nCas9, the non-complementary strand will be gradually exposed. At this time, TadA will hydrolyze and deaminate A within a certain active window on the non-complementary strand to generate inosine (I). Due to the limitation of the DNA polymerase active site, I pairs with C and is then read or replicated as G, realizing the base conversion of A-to-G (or T-to-C on the complementary strand) on the non-complementary strand.

[0004] However, existing ABE systems generally exhibit a wide targeting range and a broad editing window, and there are still deficiencies in precise editing, which need to be further optimized. Therefore, it is necessary to develop an ABE with high activity and a narrowed editing window, so as to greatly expand the application of gene editing tools, especially to provide a powerful research tool for the precise treatment of clinical diseases. Summary of the Invention

[0005] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a novel adenine base editing tool with high editing efficiency and narrow range, as well as its method and application, so as to improve the precision of existing ABE editing, narrow its editing window, and increase its application in the biomedical field.

[0006] To achieve this purpose, the present invention provides an adenine base editing tool, which is characterized in that it includes an NG-ABE9e expression vector, and the NG-ABE9e expression vector is plasmid pCMV-NG-ABE9e, and its sequence is as shown in SEQ ID NO.1. Plasmid pCMV-NG-ABE9e is used to encode and express a mutant protein: R111T+N127K+Q154R.

[0007] The adenine base editing tool includes an sgRNA expression vector. The sequence of the sgRNA expression vector is different for different gene targets.

[0008] The sgRNA expression vector is one or more of the sequences as shown in SEQ ID NOs. 2-13.

[0009] The sgRNA expression vector is as shown in SEQ ID NO.14.

[0010] The adenine base editing tool further includes eukaryotic cells and gene transfection and transduction reagents.

[0011] The amino acid sequence of NG-ABE9e encoded by the NG-ABE9e expression vector is as shown in SEQ ID NO.30.

[0012] The present invention also provides an adenine base editing tool, including the amino acid sequence of NG-ABE9e as shown in SEQ ID NO.30.

[0013] The present invention also provides a construction method of the above adenine base editing tool, which is characterized in that: using primers containing corresponding mutation information, amplifying mutation fragments one, two, three, and four from the pCMV-NG-ABE8e vector; after digesting the pCMV-NG-ABE8e vector, obtaining a backbone; after bridging mutation fragments one, two, three, and four, obtaining the expression vector pCMV-NG-ABE9e encoding the mutant protein through seamless cloning with the backbone.

[0014] The mutant fragment 1 was obtained using primer sequences containing corresponding mutation information as shown in SEQ ID NO.18 and SEQ ID NO.24; the mutant fragment 2 was obtained using primer sequences containing corresponding mutation information as shown in SEQ ID NO.23 and SEQ ID NO.20; the mutant fragment 3 was obtained using primer sequences containing corresponding mutation information as shown in SEQ ID NO.19 and SEQ ID NO.22; the mutant fragment 4 was obtained using primer sequences containing corresponding mutation information as shown in SEQ ID NO.21 and SEQ ID NO.25.

[0015] Preferably, the sequences of the mutant fragments 1, 2, 3, and 4 are as shown in SEQ ID NO.26, 27, 28, and 29.

[0016] The present invention also provides the application of the above adenine base editing tool in single-base editing of eukaryotic cells.

[0017] Preferably, the adenine base editing tool is used for the repair of eukaryotic cells containing the mutant RHO gene.

[0018] Preferably, the method for repairing eukaryotic cells containing the mutant RHO gene comprises the following steps:

[0019] (1) Design corresponding sgRNA according to the DNA target sequence of the mutRHO pathogenic gene;

[0020] (2) Inoculate eukaryotic cells containing the mutant RHO gene in a 12-well plate and adjust the cell density; after 24 h, co-transfer the sgRNA targeting the DNA target of the mutRHO pathogenic gene and the NG-ABE9e expression vector into HEK-293-mutRHO cells; replace the fresh medium after 48 h;

[0021] (3) Collect the cells on the third day and perform DNA sequencing to determine the adenine base editing result.

[0022] The present invention also provides an application method of the above novel adenine base editing tool, which is characterized in that:

[0023] (1) Construct an EGFP reporter system for detecting the editing efficiency and editing window of NG-ABE9e, and the EGFP gene sequence used is as shown in SEQ ID NO.15 and SEQ ID NO.16;

[0024] (2) Construct an NG-ABE9e disease repair model HEK-293-mutRHO cell line, and the mutRHO gene sequence used is as shown in SEQ ID NO.17;

[0025] (3) For the digested and filtered eukaryotic cells after treatment, flow cytometry analysis is performed; or the genomic DNA of the treated eukaryotic cells is extracted, PCR amplification is carried out using specific primers targeting the target, and then a high-throughput sequencing DNA library is constructed using the PCR products of the genomic DNA, and high-throughput DNA sequencing is performed.

[0026] Compared with the existing technologies, the beneficial effects of the present invention are as follows: By modifying the adenosine deaminase coding sequence of NG-ABE8e and introducing key mutation information, the editing range is successfully narrowed while maintaining its high editing efficiency. It provides new methods and ideas for achieving efficient, precise, and safe base editing. This will contribute to promoting the wider application of genome editing in basic research, clinical treatment, microorganisms, and plant and animal breeding, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a schematic diagram of the pCMV-NG-ABE9e plasmid;

[0028] Figure 2 It is a schematic diagram of the pSin-dEGFP1 / 2-IRES-Puro structure and the corresponding sgRNA sequence;

[0029] Figure 3 It is a flow chart for the establishment of the adenine base editor EGFP reporter system;

[0030] Figure 4 It is a comparison chart of the editing efficiencies of NG-ABE8e and NG-ABE9e at different A base sites;

[0031] Figure 5 It is a schematic diagram of the pSin-dEGFP1-IRES-Puro plasmid;

[0032] Figure 6 It is a schematic diagram of the pSin-dEGFP2-IRES-Puro plasmid;

[0033] Figure 7 It is a construction and repair diagram of the disease model HEK-293-mutRHO cell line;

[0034] Figure 8 It is a design diagram of sgRNA-RHO;

[0035] Figure 9 It is a repair efficiency and editing by-product diagram of NG-ABE8e and NG-ABE9e for the mutant RHO gene.

[0036] Figure 10 It is a schematic diagram of the pSin-mutRHO-EGFP-IRES-Puro plasmid. Detailed implementation manners

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the described embodiments of the present invention fall within the scope of protection of the present invention.

[0038] To further elaborate on the technical means and effects adopted by the present invention to achieve the predetermined invention objectives, the following further details the specific implementation manners, structures, features, and their effects of the present invention with reference to the accompanying drawings and preferred embodiments.

[0039] Referring to Figure 1 As shown, the present invention provides a novel adenine base editing tool, including a plasmid pCMV-NG-ABE9e:R111T+N127K+Q154R encoding and expressing an NG-ABE9e mutant protein, and its sequence is as shown in SEQ ID NO.1. The adenine base editing tool further includes an sgRNA expression vector, eukaryotic cells, and gene transfection and transduction reagents. The sequence of the sgRNA expression vector is different for different gene targets.

[0040] The present invention also provides an NG-ABE9e protein, as shown in SEQ ID NO.30.

[0041] The construction method of the above novel adenine base editing tool includes the following steps:

[0042] (1) Design primers containing corresponding mutation information (the primer sequences are as shown in SEQ ID NOs. 18-25);

[0043] (2) Amplify mutant fragments one, two, three, and four from the pCMV-NG-ABE8e vector, and the sequences are as shown in SEQ ID NOs. 26, 27, 28, and 29;

[0044] (3) Obtain a backbone after digesting the pCMV-NG-ABE8e vector with enzymes;

[0045] (4) After bridging the amplified fragments, obtain an expression vector pCMV-NG-ABE9e encoding a mutant protein by seamless cloning with the backbone.

[0046] Specifically, the sequences of SEQ ID NOs. 18-25 (synthesized artificially) are as follows

[0047] Mutant fragment one (SEQ ID NO.26) is synthesized by the following primers

[0048]

[0049] The second mutant fragment (SEQ ID NO.27) was synthesized by the following primers

[0050]

[0051] The third mutant fragment (SEQ ID NO.28) was synthesized by the following primers

[0052]

[0053] The fourth mutant fragment (SEQ ID NO.29) was synthesized by the following primers

[0054]

[0055] The application of the above novel adenine base editing tool is as follows:

[0056] (1) Construct an EGFP reporter system for detecting the editing efficiency and editing window of NG-ABE9e, which utilizes the dEGFP1 and / or dEGFP2 genes, and the sequences are as shown in SEQ ID NO.15 and 16;

[0057] (2) Construct an NG-ABE9e disease repair model HEK-293-mutRHO cell line, and the mutRHO gene sequence it utilizes is as shown in SEQ ID NO.17;

[0058] (3) Digest and filter the processed eukaryotic cells, and perform flow cytometry analysis; or extract the genomic DNA of the processed eukaryotic cells, perform PCR amplification using specific primers for the target, then construct a high-throughput sequencing DNA library using the PCR product of the genomic DNA, and perform high-throughput DNA sequencing.

[0059] The following is elaborated in specific embodiments:

[0060] Example 1: Construction of pCMV-NG-ABE9e plasmid

[0061] (1) Set corresponding mutations on the primers (the primer sequences are as shown in SEQ ID NO.18-25), and use the PCR method to amplify the sequences containing the R111T, N127K, and Q154R mutation sites on the pCMV-NG-ABE8e vector, namely the first, second, third, and fourth mutant fragments, and the sequences are as shown in SEQ ID NO.26, 27, 28, and 29.

[0062] (2) Digest the pCMV-NG-ABE8e vector with SacII and BseRI enzymes, and recover the backbone.

[0063] (3) The amplified fragments were successively ligated and then separately ligated to the backbone through In-Fusion seamless cloning technology (ClonExpressII One Step Cloning Kit) to obtain the mutant vector pCMV-NG-ABE9e, as Figure 1 shown.

[0064] (4) The PCR system used in the plasmid construction experiment is as follows:

[0065] Template: 10 ng; Forward Primer: (10 μM) 1 μL; Reverse Primer: (10 μM) 1 μL;

[0066] dNTP: 0.5 μL; DNA polymerase (Vazyme, P501): 0.5 μL; 5x Buffer: 10 μL; RNase-Water was added to make up to 50 μL.

[0067] The PCR program is as follows:

[0068] 95 °C, 3 min; 95 °C, 15 sec; 60 °C, 15 sec; 72 °C, 1 min; 35 cycles; 72 °C, 5 min.

[0069] (5) The enzyme digestion system used in the plasmid construction experiment is as follows:

[0070]

[0071]

[0072] Example 2: Establishment of an adenine base editor EGFP reporter system (as Figure 3 shown)

[0073] (1) Construct the pSin-dEGFP1 / 2-IRES-Puro lentiviral vector, which contains the dEGFP1 / 2 gene (the sequences of which are shown in SEQ ID NO. 15 and 16), IRES and puromycin resistance gene. The pSin-dEGFP1-IRES-Puro plasmid (as Figure 5 shown), the pSin-dEGFP2-IRES-Puro plasmid (as Figure 6 shown) were co-transfected into HEK-293T cells with the helper plasmids pVSVg and psPAX2 respectively. The viral supernatant was collected and filtered through a 0.45 μm filter membrane to obtain the pSin-dEGFP1-IRES-Puro lentiviral vector and the pSin-dEGFP2-IRES-Puro lentiviral vector respectively. After aliquoting, they were stored at -80 °C for later use.

[0074] (2) HEK-293 cell resuscitation: Take out the cryopreserved HEK-293 cells from liquid nitrogen, immediately place them in a 37 °C water bath, gently shake. After the liquid has completely melted (about 1 - 1.5 min), take them out, wipe and disinfect with 75% alcohol, and place them in a laminar flow hood; transfer the above cell suspension to a sterilized centrifuge tube, centrifuge at 800 rpm for 2 - 3 min; discard the supernatant, add 1 ml of complete medium to resuspend the cells. Transfer the cells to a 10 cm culture dish containing 10 ml of complete medium and gently shake to evenly distribute the cells in the culture dish; place the culture dish in a 37 °C, 5% CO₂ incubator for culture, and change the medium after the cells adhere. Preparation of complete medium: DMEM (high glucose) + 10% FBS (fetal bovine serum) + 1% Pen. / Strep. (penicillin 100 U / ml, streptomycin 100 μg / ml).

[0075] (3) Digest HEK-293 cells in the logarithmic growth phase with trypsin. After cell counting, inoculate them into a 6-well plate, and adjust the cell density to 3.0×10 5 cells / well; According to the results of lentivirus titer determination, infect with appropriate amounts of pSin-dEGFP1-IRES-Puro lentiviral vector and pSin-dEGFP2-IRES-Puro lentiviral vector respectively, and simultaneously add polybrene with a final concentration of 4 μg / ml; change the medium 12 h after lentivirus infection, and passage to a 10 cm dish 48 h later, and screen with 1 μg / ml concentration of the antibiotic Puromycin; after 10 d, pick monoclonal cells under a microscope to obtain cell lines of monoclonal HEK-293-dEGFP1 / 2 integrated with pSin-dEGFP1 / 2 genes respectively.

[0076] (4) Expand the culture of monoclonal HEK-293-dEGFP1 / 2 cells. Take some monoclonal cells to extract genes, amplify the target sequence and send it for sequencing and identification.

[0077] The helper plasmids are pVSVg and psPAX2 (preserved in the applicant's laboratory).

[0078] Lentivirus pSin-dEGFP1 / 2-IRES-Puro, stored at -80 °C.

[0079] Example 3: Determination of the editing efficiency of different A sites of pCMV-NG-ABE9e in the EGFP reporter system

[0080] (1) Design of pJET-U6-sgRNA: Design sgRNAs targeting different A sites according to the dEGFP1 / 2 gene sequence, and the sequences are shown in SEQ ID NO.2 - 13 (Figure 2 shows the designed method). As shown in the following table:

[0081]

[0082] (2) After counting the HEK-293-dEGFP1 / 2 cells, they were seeded into 12-well plates respectively, and the cell density in each well was 1.8×10 5 cells / well, and cultured in an incubator at 37°C and 5% CO2.

[0083] (3) After 24 h of cell growth, pCMV-NG-ABE8e (200 ng in the control group), pCMV-NG-ABE9e (200 ng in the experimental group) and the corresponding pJET-U6-sgRNAs (100 ng) were co-transfected into the constructed eukaryotic cell line monoclonal HEK-293-dEGFP1 / 2 cells by the transfection reagent TurboFect (Thermo Fisher, #R0531).

[0084] (4) Eukaryotic cell transfection method: After mixing the plasmids to be transfected in proportion, they were mixed into 50 μL of DMEM containing 1.5 μL of the transfection reagent TurboFect, pipetted and mixed well, and after standing at room temperature for 15 min, they were added to the 12-well plates for transfection (the transfection process is shown in the Figure 3 second half).

[0085] (5) The fresh medium was replaced 24 h after transfection, and the cells were collected 48 h later. The ratio of green fluorescent cells was detected by flow cytometry, and the editing efficiency of NG-ABE9e at different A sites and the change of the editing window were judged according to the ratio.

[0086] As Figure 4 shown, the editing efficiency of NG-ABE9e at the A6 site is comparable to that of NG-ABE8e, while it is significantly reduced at the sites after A7. Therefore, compared with NG-ABE8e, the editing window of NG-ABE9e has an obvious narrowing phenomenon.

[0087] Example 4: Construction and repair of the disease model HEK-293-mutRHO cell line (as Figure 7 , Figure 8 )

[0088] (1) Construction of the pSin-mutRHO-EGFP-IRES-Puro lentiviral vector (as Figure 10As shown in the figure, it contains the mutant RHO gene (as shown in SEQ ID NO.17), EGFP, IRES and puromycin resistance gene; the method of infecting HEK-293 cells with lentivirus is the same as that in Example 2 to obtain the HEK-293-mutRHO cell line.

[0089] (2) Design corresponding sgRNA according to the mutRHO gene sequence (as shown in SEQ ID NO.14). (As Figure 8 shown)

[0090] (3) Digest HEK-293-mutRHO cells in the logarithmic growth phase with trypsin, count them and inoculate them into a 12-well plate, and adjust the cell density to 1.8×10 5 cells / well; after 24 hours, co-transfer the sgRNA targeting the DNA target of the mutRHO pathogenic gene into HEK-293-mutRHO cells with the NG-ABE8e and NG-ABE9e expression vectors respectively; after 48 hours, replace the fresh medium, collect the cells on the third day, extract the genomic DNA of the cells, then perform PCR amplification using the specific primers targeting the target, construct a high-throughput next-generation sequencing DNA library using the PCR products of the genomic DNA, and perform high-throughput next-generation sequencing. Analysis of the next-generation data shows that the efficiency of complete repair of the mutation site by NG-ABE8e is 1.79 times that of NG-ABE8e, and the by-products generated by its editing are also significantly reduced compared with NG-ABE8e, with higher safety (as Figure 9 shown).

[0091] The above is only a preferred embodiment of the present invention, and does not impose any form of limitation on the present invention. Although the present invention has been disclosed above with a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to the equivalent embodiments by using the above-disclosed technical content within the scope of the technical solution of the present invention. However, as long as it does not depart from the content of the technical solution of the present invention, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention. Sequence Listing <110> Wenzhou Medical University <120> An Adenine Base Editing Tool and Its Method and Application <160> 30 <170> SIPOSequenceListing 1.0 <210> 1 <211> 8217 <212> DNA <213> Artificial Sequence <400> 1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 60 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 120 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 180 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 240 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 300 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 360 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gaaacggaca 420 gccgacggaa gcgagttcga gtcaccaaag aagaagcgga aagtctctga ggtggagttt 480 tcccacgagt actggatgag acatgccctg accctggcca agagggcacg ggatgagagg 540 gaggtgcctg tgggagccgt gctggtgctg aacaatagag tgatcggcga gggctggaac 600 agagccatcg gcctgcacga cccaacagcc catgccgaaa ttatggccct gagacagggc 660 ggcctggtca 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ccagggcgat agcctgcacg agcacattgc caatctggcc 3240 ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg 3300 aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag 3360 accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc 3420 aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac 3480 gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg 3540 gacatcaacc ggctgtccga ctacgatgtg gaccatatcg tgcctcagag ctttctgaag 3600 gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac 3660 aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac 3720 gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg 3780 agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca 3840 aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag 3900 ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag 3960 gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac 4020 ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc 4080 gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa 4140 atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc 4200 gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa 4260 accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc 4320 atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag 4380 tctatcaggc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct 4440 aagaagtacg gcggcttcgt cagccccacc gtggcctatt ctgtgctggt ggtggccaaa 4500 gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc 4560 atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa 4620 gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac 4680 ggccggaaga gaatgctggc ctctgccaga ttcctgcaga agggaaacga actggccctg 4740 ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc 4800 cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag 4860 atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac 4920 aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat 4980 atcatccacc tgtttaccct gaccaatctg ggagccccta gggccttcaa gtactttgac 5040 accaccatcg accggaaggt gtacaggagc accaaagagg tgctggacgc caccctgatc 5100 caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggtgac 5160 tctggcggct caaaaagaac cgccgacggc agcgaattcg agcccaagaa gaagaggaaa 5220 gtctaaccgg tcatcatcac catcaccatt gagtttaaac ccgctgatca gcctcgactg 5280 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg 5340 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga 5400 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg 5460 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa 5520 ccagctgggg ctcgataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc 5580 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca 5640 taaagtgtaa agcctagggt gcctaatgag tgagctaact cacattaatt gcgttgcgct 5700 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac 5760 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc 5820 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 5880 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 5940 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 6000 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 6060 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 6120 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 6180 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 6240 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 6300 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 6360 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag 6420 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 6480 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 6540 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacactc 6600 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 6660 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 6720 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 6780 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct 6840 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt 6900 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 6960 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 7020 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg 7080 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt 7140 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg 7200 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg 7260 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc 7320 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa 7380 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac 7440 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt 7500 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg 7560 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa 7620 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata 7680 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg 7740 gagatcgatc tcccgatccc ctagggtcga ctctcagtac aatctgctct gatgccgcat 7800 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca 7860 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg 7920 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat 7980 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatattgagt 8040 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc 8100 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac 8160 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatc 8217 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 ctagccctcg ctggtgtcgt 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 gccctagccc tcgctggtgt 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 cgtgccctag ccctcgctgg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 cccgtgccct agccctcgct 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <400> 6 gcccgtgccc tagccctcgc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <400> 7 gctgcccgtg ccctagccct 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <400> 8 accctcgctg gtgtcgtcgc 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <400> 9 ctgaccctcg ctggtgtcgt 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <400> 10 gccctgaccc tcgctggtgt 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <400> 11 cgtgccctga ccctcgctgg 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <400> 12 gcccgtgccc tgaccctcgc 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <400> 13 gctgcccgtg ccctgaccct 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <400> 14 cgcccatcgc attggagaag 20 <210> 15 <211> 738 <212> DNA <213> Artificial Sequence <400> 15 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctgaccctcg 180 ctggtgtcgt cgctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtcc 720 ggactcagat ctcgataa 738 <210> 16 <211> 738 <212> DNA <213> Artificial Sequence <400> 16 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctgaccctcg 180 ctggtgtcgt cgctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtcc 720 ggactcagat ctcgataa 738 <210> 17 <211> 78 <212> DNA <213> Artificial Sequence <400> 17 atgaatggca cagaaggccc taacttctac gtgcccttct ccaatgcgat gggcgtggtg 60 cggagcccct tcgagcag 78 <210> 18 <211> 23 <212> DNA <213> Artificial Sequence <400> 18 tcagatccgc tagagatccg cgg 23 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <400> 19 taccccggca tgaagcaccg c 21 <210> 20 <211> 22 <212> DNA <213> Artificial Sequence <400> 20 gtgcttcatg ccggggtagt tc 22 <210> 21 <211> 30 <212> DNA <213> Artificial Sequence <400> 21 tagacgggtg ttcaatgctc agaagaaggc 30 <210> 22 <211> 36 <212> DNA <213> Artificial Sequence <400> 22 ttgaacaccc gtctaggcat ccgatagaaa tcgcac 36 <210> 23 <211> 23 <212> DNA <213> Artificial Sequence <400> 23 ctcaaaaacc ggcgccgcag gct 23 <210> 24 <211> 32 <212> DNA <213> Artificial Sequence <400> 24 gcgccggttt ttgagttcct cacgccaaac ac 32 <210> 25 <211> 27 <212> DNA <213> Artificial Sequence <400> 25 tcagagccag aggagcctcc gctagat 27 <210> 26 <211> 451 <212> DNA <213> Artificial Sequence <400> 26 tcagatccgc tagagatccg cggccgctaa tacgactcac tatagggaga gccgccacca 60 tgaaacggac agccgacgga agcgagttcg agtcaccaaa gaagaagcgg aaagtctctg 120 aggtggagtt ttcccacgag tactggatga gacatgccct gaccctggcc aagagggcac 180 gggatgagag ggaggtgcct gtgggagccg tgctggtgct gaacaataga gtgatcggcg 240 agggctggaa cagagccatc ggcctgcacg acccaacagc ccatgccgaa attatggccc 300 tgagacaggg cggcctggtc atgcagaact acagactgat tgacgccacc ctgtacgtga 360 cattcgagcc ttgcgtgatg tgcgccggcg ccatgatcca ctctaggatc ggccgcgtgg 420 tgtttggcgt gaggaactca aaaaccggcg c 451 <210> 27 <211> 61 <212> DNA <213> Artificial Sequence <400> 27 ctcaaaaacc ggcgccgcag gctccctgat gaacgtgctg aactaccccg gcatgaagca 60 c 61 <210> 28 <211> 105 <212> DNA <213> Artificial Sequence <400> 28 taccccggca tgaagcaccg cgtcgaaatt accgagggaa tcctggcaga tgaatgtgcc 60 gccctgctgt gcgatttcta tcggatgcct agacgggtgt tcaat 105 <210> 29 <211> 81 <212> DNA <213> Artificial Sequence <400> 29 tagacgggtg ttcaatgctc agaagaaggc ccagagctcc atcaactccg gaggatctag 60 cggaggctcc tctggctctg a 81 <210> 30 <211> 1605 <212> PRT <213> Artificial Sequence <400> 30 Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys 1 5 10 15 Arg Lys Val Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His 20 25 30 Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val 35 40 45 Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn 50 55 60 Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala 65 70 75 80 Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala 85 90 95 Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met 100 105 110 Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ser Lys 115 120 125 Thr Gly Ala Ala Gly Ser Leu Met Asn Val Leu Asn Tyr Pro Gly Met 130 135 140 Lys His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala 145 150 155 160 Ala Leu Leu Cys Asp Phe Tyr Arg Met Pro Arg Arg Val Phe Asn Ala 165 170 175 Gln Lys Lys Ala Gln Ser Ser Ile Asn Ser Gly Gly Ser Ser Gly Gly 180 185 190 Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu 195 200 205 Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys Tyr Ser Ile Gly 210 215 220 Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu 225 230 235 240 Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg 245 250 255 His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly 260 265 270 Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr 275 280 285 Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn 290 295 300 Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser 305 310 315 320 Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly 325 330 335 Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr 340 345 350 His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg 355 360 365 Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe 370 375 380 Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu 385 390 395 400 Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro 405 410 415 Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu 420 425 430 Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu 435 440 445 Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu 450 455 460 Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu 465 470 475 480 Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala 485 490 495 Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu 500 505 510 Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile 515 520 525 Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His 530 535 540 His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro 545 550 555 560 Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala 565 570 575 Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile 580 585 590 Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys 595 600 605 Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly 610 615 620 Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg 625 630 635 640 Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile 645 650 655 Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala 660 665 670 Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr 675 680 685 Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala 690 695 700 Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn 705 710 715 720 Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val 725 730 735 Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys 740 745 750 Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu 755 760 765 Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr 770 775 780 Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu 785 790 795 800 Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile 805 810 815 Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu 820 825 830 Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile 835 840 845 Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met 850 855 860 Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg 865 870 875 880 Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu 885 890 895 Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu 900 905 910 Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln 915 920 925 Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala 930 935 940 Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val 945 950 955 960 Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val 965 970 975 Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn 980 985 990 Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly 995 1000 1005 Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn 1010 1015 1020 Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val 1025 1030 1035 1040 Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His 1045 1050 1055 Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val 1060 1065 1070 Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser 1075 1080 1085 Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn 1090 1095 1100 Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu 1105 1110 1115 1120 Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln 1125 1130 1135 Leu Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp 1140 1145 1150 Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu 1155 1160 1165 Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys 1170 1175 1180 Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala 1185 1190 1195 1200 His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys 1205 1210 1215 Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr 1220 1225 1230 Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala 1235 1240 1245 Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr 1250 1255 1260 Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu 1265 1270 1275 1280 Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe 1285 1290 1295 Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys 1300 1305 1310 Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Arg Pro 1315 1320 1325 Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1330 1335 1340 Lys Lys Tyr Gly Gly Phe Val Ser Pro Thr Val Ala Tyr Ser Val Leu 1345 1350 1355 1360 Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val 1365 1370 1375 Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys 1380 1385 1390 Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys 1395 1400 1405 Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn 1410 1415 1420 Gly Arg Lys Arg Met Leu Ala Ser Ala Arg Phe Leu Gln Lys Gly Asn 1425 1430 1435 1440 Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser 1445 1450 1455 His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln 1460 1465 1470 Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln 1475 1480 1485 Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp 1490 1495 1500 Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu 1505 1510 1515 1520 Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala 1525 1530 1535 Pro Arg Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Val Tyr 1540 1545 1550 Arg Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile 1555 1560 1565 Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1570 1575 1580 Ser Gly Gly Ser Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Pro Lys 1585 1590 1595 1600 Lys Lys Arg Lys Val 1605

Claims

1. An adenine base editing tool, characterized in that, Comprising an NG-ABE9e expression vector, the sequence of which is shown in SEQ ID NO.

1.

2. The adenine base editing tool according to claim 1, wherein Comprising an sgRNA expression vector.

3. The adenine base editing tool according to claim 2, characterized in that, An sgRNA expression vector, the sequence of which is shown in SEQ ID NO.

14.

4. The adenine base editing tool according to any one of claims 1-3, characterized in that, The amino acid sequence of NG-ABE9e encoded by the NG-ABE9e expression vector is shown in SEQ ID NO.

30.

5. An adenine base editing tool, characterized in that, Comprising an amino acid sequence of NG-ABE9e, which is shown in SEQ ID NO.

30.

6. The method for constructing an adenine base editing tool according to any one of claims 1-4, characterized in that : Using primers containing corresponding mutation information, amplify mutant fragments one, two, three, and four from the NG-ABE8e vector; after digesting the NG-ABE8e vector, obtain the backbone; after bridging mutant fragments one, two, three, and four, obtain the NG-ABE9e expression vector encoding the mutant protein by seamless cloning with the backbone.

7. The method for constructing an adenine base editing tool according to claim 6, wherein, Obtain mutant fragment one using the primer sequences containing corresponding mutation information shown in SEQ ID NO.18 and SEQ ID NO.24; obtain mutant fragment two using the primer sequences containing corresponding mutation information shown in SEQ ID NO.23 and SEQ ID NO.20; obtain mutant fragment three using the primer sequences containing corresponding mutation information shown in SEQ ID NO.19 and SEQ ID NO.22; obtain mutant fragment four using the primer sequences containing corresponding mutation information shown in SEQ ID NO.21 and SEQ ID NO.

25.

8. The method for constructing the adenine base editing tool according to claim 7, wherein The sequences of the mutant fragments one, two, three, and four are shown in SEQ ID NO.26, 27, 28, and 29.

9. Use of an adenine base editing tool according to any one of claims 1-5 in eukaryotic cell single base editing.

10. Use of an adenine base editing tool as described in claim 9, characterized in that: The adenine base editing tool is used for the repair of eukaryotic cells containing a mutant RHO gene.

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