A recombinant glycosylase base editing system and its application

Abstract

The present invention discloses a recombinant glycosylase base editing system and its application. The recombinant glycosylase base editing system of the present invention is an APOBEC-nCas9-Ung1 or APOBEC(R33A)-nCas9-Rad51-Ung1 base editing system; the base editing system APOBEC-nCas9-Ung1 includes cytosine deaminase APOBEC, nuclease nCas9 and uracil DNA glycosylase Ung1; the base editing system APOBEC(R33A)-nCas9-Rad51-Ung1 includes cytosine deaminase APOBEC(R33A), nuclease nCas9, single-stranded DNA binding protein Rad51 and uracil DNA glycosylase Ung1. Experiments have shown that the recombinant glycosylase base editing system of the present invention can improve the editing efficiency of base C to base G in the genome sequence of an organism or a biological cell and the purity of the editing product.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a recombinant glycosylase base editing system and an application thereof, and in particular to a recombinant glycosylase base editing system capable of improving C to G base editing efficiency and product purity and an application thereof in base editing. Background Art

[0002] Based on the CRISPR / Cas9 system, Liu Ruqian's research group at Harvard University reported two new DNA base editing systems that can act on single bases - the cytosine base editing system (CBE) and the adenine base editing system (ABE), which can respectively achieve efficient conversion of cytosine (C) to thymine (T) and adenine (A) to guanine (G). Later, Li Dali's research group at East China Normal University developed a new dual-functional base editing system A&C-BEmax, which can not only achieve single C to T or A to G, but also achieve efficient conversion of C to T and A to G on the same allele. At the same time, J. Keith Joung's team reported a synchronous programmable adenine and cytosine editing system (SPACE) based on CRISPR / Cas9, which can simultaneously perform A to G and C to T editing.

[0003] Following the CBE, ABE, A&C-Bemax and SPACE base editing systems, another highly anticipated specific base editing system, the novel glycosylase base editing system (GBE), has been developed. The development of this base editing system has made base transversion a reality. The GBE base editing system consists of three components: APOBEC, Cas9-nickase (nCas9) and uracil DNA glycosylase (Ung). Its editing strategy is as follows: Under the action of cytosine deaminase (APOBEC), cytosine (C) on the DNA single strand is converted to uracil (U), and uracil DNA glycosylase (Ung) removes U at this site to establish an apurinic / apyrimidinic (AP) site. The DNA damage site induces the initiation of DNA repair, thereby achieving base transversion.

[0004] The GBE base editing system mainly converts the 6th cytosine (C) of the pre-spacer sequence to guanine (G), and realizes C to G base editing at the C6 position. Although the GBE base editing system can realize C to G base conversion in mammalian cells, the base editing system is still imperfect, and there are problems such as low base editing efficiency and low purity of C to G products. In order to better apply GBE technology, it is very important to improve the performance of GBE technology. Therefore, it is urgent to optimize the existing GBE base editing system and develop a recombinant glycosylase base editing system with high base editing efficiency and pure C to G products. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to improve the base editing efficiency of the GBE base editing system and the purity of the base editing products.

[0006] In order to solve the above technical problems, the present invention first provides a base editing system APOBEC-nCas9-Ung1 or a base editing system APOBEC(R33A)-nCas9-Rad51-Ung1;

[0007] The base editing system APOBEC-nCas9-Ung1 includes cytosine deaminase APOBEC or biological materials related thereto, nuclease nCas9 or biological materials related thereto, and uracil DNA glycosylase Ung1 or biological materials related thereto;

[0008] The base editing system APOBEC (R33A)-nCas9-Rad51-Ung1 includes cytosine deaminase APOBEC (R33A) or biological materials related thereto, nuclease nCas9 or biological materials related thereto, single-stranded DNA binding protein Rad51 or biological materials related thereto, and uracil DNA glycosylase Ung1 or biological materials related thereto.

[0009] In the above base editing system, the cytosine deaminase APOBEC is A1) or A2) or A3):

[0010] A1) The amino acid sequence is the protein shown in SEQ ID NO: 1;

[0011] A2) a protein having the same function as the amino acid sequence shown in SEQ ID NO: 1 in the sequence list, wherein one or more amino acid residues are substituted and / or deleted and / or added;

[0012] A3) a fusion protein obtained by connecting a tag to the N-terminus or / and the C-terminus of A1) or A2);

[0013] The biological material related to the cytosine deaminase APOBEC is any one of B1) to B5):

[0014] B1) a nucleic acid molecule encoding the cytosine deaminase APOBEC;

[0015] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0016] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0017] B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0018] B5) A transgenic cell line containing the nucleic acid molecule described in B1), or a transgenic cell line containing the expression cassette described in B2), or a transgenic cell line containing the recombinant vector described in B3).

[0019] The nuclease nCas9 is C1) or C2) or C3):

[0020] C1) The amino acid sequence is the protein shown in SEQ ID NO: 2;

[0021] C2) a protein having the same function as the amino acid sequence shown in Sequence 2 in the sequence list, wherein one or more amino acid residues are substituted and / or deleted and / or added;

[0022] C3) A fusion protein obtained by connecting a tag to the N-terminus or / and the C-terminus of C1) or C2);

[0023] The biological material associated with the nuclease nCas9 is any one of D1) to D5):

[0024] D1) a nucleic acid molecule encoding the nuclease nCas9;

[0025] D2) an expression cassette containing the nucleic acid molecule described in D1);

[0026] D3) a recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2);

[0027] D4) a recombinant microorganism containing the nucleic acid molecule described in D1), or a recombinant microorganism containing the expression cassette described in D2), or a recombinant microorganism containing the recombinant vector described in D3);

[0028] D5) A transgenic cell line containing the nucleic acid molecule described in D1), or a transgenic cell line containing the expression cassette described in D2), or a transgenic cell line containing the recombinant vector described in D3).

[0029] The uracil DNA glycosylase Ung1 is E1) or E2) or E3):

[0030] E1) The amino acid sequence is the protein shown in SEQ ID NO: 3;

[0031] E2) a protein having the same function as the amino acid sequence shown in Sequence 3 in the sequence list, wherein one or more amino acid residues are substituted and / or deleted and / or added;

[0032] E3) A fusion protein obtained by connecting a tag to the N-terminus or / and C-terminus of E1) or E2);

[0033] The biological material related to the uracil DNA glycosylase Ung1 is any one of F1) to F5):

[0034] F1) a nucleic acid molecule encoding the uracil DNA glycosylase Ung1;

[0035] F2) an expression cassette containing the nucleic acid molecule described in F1);

[0036] F3) a recombinant vector containing the nucleic acid molecule described in F1), or a recombinant vector containing the expression cassette described in F2);

[0037] F4) a recombinant microorganism containing the nucleic acid molecule described in F1), or a recombinant microorganism containing the expression cassette described in F2), or a recombinant microorganism containing the recombinant vector described in F3);

[0038] F5) A transgenic cell line containing the nucleic acid molecule of F1), or a transgenic cell line containing the expression cassette of F2), or a transgenic cell line containing the recombinant vector of F3).

[0039] The cytosine deaminase APOBEC (R33A) is G1) or G2) or G3):

[0040] G1) The amino acid sequence is the protein shown in SEQ ID NO:4;

[0041] G2) a protein having the same function as the amino acid sequence shown in Sequence 4 in the sequence list, wherein one or more amino acid residues are substituted and / or deleted and / or added;

[0042] G3) A fusion protein obtained by connecting a tag to the N-terminus or / and the C-terminus of G1) or G2);

[0043] The biological material related to the cytosine deaminase APOBEC (R33A) is any one of H1) to H5):

[0044] H1) a nucleic acid molecule encoding the cytosine deaminase APOBEC (R33A);

[0045] H2) an expression cassette containing the nucleic acid molecule described in H1);

[0046] H3) a recombinant vector containing the nucleic acid molecule described in H1), or a recombinant vector containing the expression cassette described in H2);

[0047] H4) a recombinant microorganism containing the nucleic acid molecule described in H1), or a recombinant microorganism containing the expression cassette described in H2), or a recombinant microorganism containing the recombinant vector described in H3);

[0048] H5) A transgenic cell line containing the nucleic acid molecule described in H1), or a transgenic cell line containing the expression cassette described in H2), or a transgenic cell line containing the recombinant vector described in H3).

[0049] The single-stranded DNA binding protein Rad51 is I1) or I2) or I3):

[0050] I1) the amino acid sequence is the protein shown in SEQ ID NO: 5;

[0051] I2) a protein having the same function as the amino acid sequence shown in Sequence 5 in the sequence list, wherein one or more amino acid residues are substituted and / or deleted and / or added;

[0052] I3) A fusion protein obtained by connecting a tag to the N-terminus or / and the C-terminus of I1) or I2);

[0053] The biological material related to the single-stranded DNA binding protein Rad51 is any one of J1) to J5):

[0054] J1) a nucleic acid molecule encoding the single-stranded DNA binding protein Rad51;

[0055] J2) an expression cassette containing the nucleic acid molecule described in J1);

[0056] J3) a recombinant vector containing the nucleic acid molecule described in J1), or a recombinant vector containing the expression cassette described in J2);

[0057] J4) a recombinant microorganism containing the nucleic acid molecule described in J1), or a recombinant microorganism containing the expression cassette described in J2), or a recombinant microorganism containing the recombinant vector described in J3);

[0058] J5) A transgenic cell line containing the nucleic acid molecule described in J1), or a transgenic cell line containing the expression cassette described in J2), or a transgenic cell line containing the recombinant vector described in J3).

[0059] In order to facilitate the expression, detection, tracing and / or purification of the proteins in A1), C1), E1), G1) or I1) above, a tag may be connected to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in Sequence 1 or Sequence 2 or Sequence 3 or Sequence 4 or Sequence 5 in the sequence listing, and the tag includes but is not limited to: Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag and / or SUMO tag.

[0060] The protein in A1), C1), E1), G1) or I1) is a protein having 75% or more identity with the amino acid sequence of the protein shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 and having the same function. The 75% or more identity means 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0061] The proteins in A1), C1), E1), G1) or I1) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0062] The coding gene of the protein in the above A1), C1), E1), G1) or I1) can be obtained by deleting one or more codons of amino acid residues in the DNA sequence shown in SEQ ID NO: 4695-5375, SEQ ID NO: 5430-9530, SEQ ID NO: 7, SEQ ID NO: 5109-5789 or SEQ ID NO: 10089-10430, and / or performing missense mutation of one or more base pairs, and / or connecting the coding sequence of the tag shown in the above table to its 5′ end and / or 3′ end. The DNA sequences shown in SEQ ID NO: 4695-5375, SEQ ID NO: 5430-9530, SEQ ID NO: 7, SEQ ID NO: 5109-5789 and SEQ ID NO: 10089-10430 respectively encode the proteins shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.

[0063] Furthermore, the nucleic acid molecule in B1) is b1) or b2) or b3):

[0064] b1) cDNA molecules or DNA molecules shown in SEQ ID NOs. 4695-5375 of SEQ ID NO. 6 in the sequence listing;

[0065] b2) a cDNA molecule or a DNA molecule that has 75% or more identity with the nucleotide sequence defined in b1) and encodes the cytosine deaminase APOBEC;

[0066] b3) a cDNA molecule or a DNA molecule which hybridizes with the nucleotide sequence defined in b1) or b2) under stringent conditions and encodes the cytosine deaminase APOBEC.

[0067] D1) The nucleic acid molecule is d1) or d2) or d3):

[0068] d1) the cDNA molecule or DNA molecule shown in positions 5430-9530 of Sequence 6 in the sequence listing;

[0069] d2) a cDNA molecule or a DNA molecule that has 75% or more identity with the nucleotide sequence defined in d1) and encodes the nuclease nCas9;

[0070] d3) a cDNA molecule or a DNA molecule that hybridizes with the nucleotide sequence defined in d1) or d2) under stringent conditions and encodes the nuclease nCas9.

[0071] F1) The nucleic acid molecule is f1) or f2) or f3):

[0072] f1) cDNA molecule or DNA molecule shown in Sequence 7 in the sequence listing;

[0073] f2) a cDNA molecule or a DNA molecule that has 75% or more identity with the nucleotide sequence defined in f1) and encodes the uracil DNA glycosylase Ung1;

[0074] f3) a cDNA molecule or a DNA molecule which hybridizes with the nucleotide sequence defined in f1) or f2) under stringent conditions and encodes the uracil DNA glycosylase Ung1.

[0075] H1) The nucleic acid molecule is h1) or h2) or h3):

[0076] h1) the cDNA molecule or DNA molecule shown in positions 5109-5789 of SEQ ID NO: 9 in the sequence listing;

[0077] h2) a cDNA molecule or a DNA molecule that has 75% or more identity with the nucleotide sequence defined in h1) and encodes the cytosine deaminase APOBEC (R33A);

[0078] h3) a cDNA molecule or a DNA molecule which hybridizes with the nucleotide sequence defined in h1) or h2) under stringent conditions and encodes the cytosine deaminase APOBEC (R33A).

[0079] J1) The nucleic acid molecule is j1) or j2) or j3):

[0080] j1) cDNA molecule or DNA molecule shown in Sequence 7 in the sequence listing;

[0081] j2) a cDNA molecule or a DNA molecule that has 75% or more identity with the nucleotide sequence defined in j1) and encodes the single-stranded DNA binding protein Rad51;

[0082] j3) a cDNA molecule or a DNA molecule which hybridizes with the nucleotide sequence defined in j1) or j2) under stringent conditions and encodes the single-stranded DNA binding protein Rad51.

[0083] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0084] A person skilled in the art can easily adopt known methods, such as directed evolution and point mutation methods, to mutate the nucleotide sequences encoding the cytosine deaminase APOBEC, the nuclease nCas9, the uracil DNA glycosylase Ung1, the cytosine deaminase APOBEC (R33A) and the single-stranded DNA binding protein Rad51 of the present invention. Those artificially modified nucleotides having 75% or higher identity with the nucleotide sequences of the cytosine deaminase APOBEC, the nuclease nCas9, the uracil DNA glycosylase Ung1, the cytosine deaminase APOBEC (R33A) and the single-stranded DNA binding protein Rad51 of the present invention, as long as they encode the cytosine deaminase APOBEC, the nuclease nCas9, the uracil DNA glycosylase Ung1, the cytosine deaminase APOBEC (R33A) and the single-stranded DNA binding protein Rad51 and have the same function, are all derived from the nucleotide sequences of the present invention and are equivalent to the sequences of the present invention.

[0085] The term "identity" as used herein refers to sequence similarity with a natural nucleic acid sequence. "Identity" includes nucleotide sequences with 75% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of the protein composed of the amino acid sequence shown in the coding sequence 1 or sequence 2 or sequence 3 or sequence 4 or sequence 5 of the present invention. Identity can be evaluated by the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. The 75% or more identity is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0086] The stringent conditions are hybridization and washing the membrane twice at 68°C in a 2×SSC, 0.1% SDS solution for 5 min each time, and hybridization and washing the membrane twice at 68°C in a 0.5×SSC, 0.1% SDS solution for 15 min each time; or hybridization and washing the membrane at 65°C in a 0.1×SSPE (or 0.1×SSC), 0.1% SDS solution.

[0087] The expression cassette refers to a DNA capable of expressing the cytosine deaminase APOBEC, the nuclease nCas9, the uracil DNA glycosylase Ung1, the cytosine deaminase APOBEC (R33A) or the single-stranded DNA binding protein Rad51 in a host cell, and the DNA may include not only a promoter for initiating transcription of the cytosine deaminase APOBEC gene, the nuclease nCas9 gene, the uracil DNA glycosylase Ung1 gene, the cytosine deaminase APOBEC (R33A) gene or the single-stranded DNA binding protein Rad51 gene, but also a terminator for terminating transcription of the cytosine deaminase APOBEC gene, the nuclease nCas9 gene, the uracil DNA glycosylase Ung1 gene, the cytosine deaminase APOBEC (R33A) gene or the single-stranded DNA binding protein Rad51 gene. Further, the expression cassette may also include an enhancer sequence.

[0088] The vector may be a plasmid, cosmid, phage or viral vector.

[0089] The microorganism may be a yeast, a bacterium, an algae or a fungus.

[0090] The cell line may be a prokaryotic cell line or a eukaryotic cell line.

[0091] Furthermore, the above-mentioned base editing system also includes gRNA or biological materials related thereto.

[0092] The gRNA is as shown in Formula I: RNA-gRNA backbone transcribed by spacer sequence (Formula I);

[0093] The gRNA backbone is an RNA molecule obtained by replacing T in positions 342-417 of sequence 8 with U.

[0094] The biological material associated with the gRNA shown in Formula I is any one of K1) to K5):

[0095] K1) a nucleic acid molecule that transcribes the gRNA shown in formula I;

[0096] K2) an expression cassette containing the nucleic acid molecule described in K1);

[0097] K3) a recombinant vector containing the nucleic acid molecule described in K1), or a recombinant vector containing the expression cassette described in K2);

[0098] K4) a recombinant microorganism containing the nucleic acid molecule described in K1), or a recombinant microorganism containing the expression cassette described in K2), or a recombinant microorganism containing the recombinant vector described in K3);

[0099] K5) A transgenic cell line containing the nucleic acid molecule described in K1) or a transgenic cell line containing the expression cassette described in K2).

[0100] In order to solve the above-mentioned technical problems, the present invention also provides new uses of the above-mentioned base editing system.

[0101] The present invention provides the use of the above-mentioned base editing system in any one of X1)-X6):

[0102] X1) Base editing of the genome sequence of an organism or biological cell;

[0103] X2) preparing products of base editing of the genome sequence of an organism or biological cell;

[0104] X3) Improving the base editing efficiency of the genome sequence of an organism or biological cell;

[0105] X4) preparing products that improve the efficiency of base editing of genomic sequences of organisms or biological cells;

[0106] X5) Improving the purity of base editing products of the genome sequence of an organism or biological cell;

[0107] X6) preparing products that improve the purity of base editing products of the genome sequence of an organism or biological cell;

[0108] The base editing is from base C to base G.

[0109] In order to solve the above technical problems, the present invention finally provides any of the following methods Y1)-Y5):

[0110] Y1) A method for base editing of a genomic sequence, comprising the following steps: introducing the above-mentioned cytosine deaminase APOBEC, nuclease nCas9, uracil DNA glycosylase Ung1 and gRNA into an organism or biological cell to achieve base editing;

[0111] Y2) A method for base editing of a genomic sequence, comprising the following steps: introducing the above-mentioned cytosine deaminase APOBEC (R33A), nuclease nCas9, single-stranded DNA binding protein Rad51, uracil DNA glycosylase Ung1 and gRNA into an organism or biological cell to achieve base editing;

[0112] Y3) A method for improving the base editing efficiency of a genome sequence of an organism or a biological cell, comprising the following steps: introducing the above-mentioned cytosine deaminase APOBEC, nuclease nCas9, uracil DNA glycosylase Ung1 and gRNA into an organism or a biological cell to achieve improved base editing efficiency;

[0113] Y4) A method for improving the base editing efficiency of a genome sequence of an organism or a biological cell, comprising the following steps: introducing the above-mentioned cytosine deaminase APOBEC (R33A), nuclease nCas9, single-stranded DNA binding protein Rad51, uracil DNA glycosylase Ung1 and gRNA into an organism or a biological cell to achieve improved base editing efficiency;

[0114] Y5) A method for improving the purity of a base editing product of a genome sequence of an organism or a biological cell, comprising the following steps: introducing the above-mentioned cytosine deaminase APOBEC (R33A), nuclease nCas9, single-stranded DNA binding protein Rad51, uracil DNA glycosylase Ung1 and gRNA into an organism or a biological cell to improve the purity of the base editing product;

[0115] The base editing is from base C to base G.

[0116] In any of the above methods, in Y1) and Y3), the method of introducing the above cytosine deaminase APOBEC, nuclease nCas9, uracil DNA glycosylase Ung1 and gRNA into an organism or biological cell is to introduce the above cytosine deaminase APOBEC coding gene, nuclease nCas9 coding gene, uracil DNA glycosylase Ung1 coding gene and gRNA transcription DNA molecule into an organism or biological cell. Further, the cytosine deaminase APOBEC coding gene, the nuclease nCas9 coding gene, the uracil DNA glycosylase Ung1 coding gene and gRNA transcription DNA molecule can be introduced into an organism or biological cell through one or more recombinant expression vectors. In a specific embodiment of the present invention, the coding gene of the cytosine deaminase APOBEC, the coding gene of the nuclease nCas9 and the coding gene of the uracil DNA glycosylase Ung1 are introduced into the biological cell through the APOBEC-nCas9-Ung1 recombinant expression vector; the DNA molecule for transcribing gRNA is introduced into the biological cell through the gRNA expression vector.

[0117] In the above Y2), Y4), and Y5), the method for introducing the above cytosine deaminase APOBEC (R33A), nuclease nCas9, single-stranded DNA binding protein Rad51, uracil DNA glycosylase Ung1, and gRNA into an organism or biological cell is to introduce the above cytosine deaminase APOBEC (R33A) coding gene, nuclease nCas9 coding gene, single-stranded DNA binding protein Rad51 coding gene, uracil DNA glycosylase Ung1 coding gene, and gRNA transcription DNA molecule into an organism or biological cell. Further, the cytosine deaminase APOBEC (R33A) coding gene, nuclease nCas9 coding gene, single-stranded DNA binding protein Rad51 coding gene, uracil DNA glycosylase Ung1 coding gene, and gRNA transcription DNA molecule can be introduced into an organism or biological cell through one or more recombinant expression vectors. In a specific embodiment of the present invention, the coding gene of cytosine deaminase APOBEC (R33A), the coding gene of nuclease nCas9, the coding gene of single-stranded DNA binding protein Rad51 and the coding gene of uracil DNA glycosylase Ung1 are introduced into biological cells through APOBEC (R33A)-nCas9-Rad51-Ung1 recombinant expression vector; the DNA molecule for transcribing gRNA is introduced into biological cells through the gRNA expression vector.

[0118] In any of the above methods, the gRNA expression vector can be specifically a RNF2-gRNA recombinant expression vector, a VISTA hs267-gRNA recombinant expression vector, RP11-177B4-1-gRNA recombinant expression vector, RP1-97D16-gRNA recombinant expression vector, PSMB2-1-gRNA recombinant expression vector, RP11-177B4-3-gRNA recombinant expression vector, RP11-582C12-2-gRNA recombinant expression vector, HIRA-1-gRNA recombinant expression vector, PPP1R12C-site3-gRNA recombinant expression vector, RP11-399K21-2-gRNA recombinant expression vector, FANCY-gRNA recombinant expression vector, FANCF-site4-gRNA recombinant expression vector, DNMT3B-OTS1-gRNA recombinant expression vector, HIRA-2-gRNA recombinant expression vector, VEGFA-gRNA recombinant expression vector, EMX1-site7-gRNA recombinant expression vector, RP11-399K21-1-gRNA recombinant expression vector.

[0119] In any of the above applications or methods, the organism may be a plant or an animal; the biological cell may be a plant cell or an animal cell. Further, the animal cell may be a mammalian cell. Further, the mammalian cell may be a HEK293T cell, a HepG2 cell or a HeLa cell.

[0120] In any of the above applications or methods, the base C to base G specifically refers to base C to base G at positions C3, C4, C5, C6, C7, and C8, wherein the C3 position is the third position from the 5' end of the pre-spacer sequence, the C4 position is the fourth position from the 5' end of the pre-spacer sequence, the C5 position is the fifth position from the 5' end of the pre-spacer sequence, the C6 position is the sixth position from the 5' end of the pre-spacer sequence, the C7 position is the seventh position from the 5' end of the pre-spacer sequence, and the C8 position is the eighth position from the 5' end of the pre-spacer sequence. Preferably, the base C to base G at position C6.

[0121] The present invention provides two base editing systems, APOBEC-nCas9-Ung1 and APOBEC (R33A) -nCas9-Rad51-Ung1. Experiments have shown that compared with the base editing system APOBEC-nCas9-Ung, the base editing system APOBEC-nCas9-Ung1 provided by the present invention can significantly improve the editing efficiency of base C to base G, and the base editing system APOBEC (R33A) -nCas9-Rad51-Ung1 can not only improve the editing efficiency of base C to base G, but also improve the purity of the editing product of base C to base G. The base editing system of the present invention has broad application prospects in base editing or replacement of plant or animal genomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0122] Figure 1 Schematic diagram of the base editing systems APOBEC-nCas9-Ung, APOBEC-nCas9-Ung1 and APOBEC(R33A)-nCas9-Rad51-Ung1.

[0123] Figure 2 The C to G base editing efficiency of the base editing systems APOBEC-nCas9-Ung, APOBEC-nCas9-cgUng, and APOBEC-nCas9-Ung1 with different Ung sources in HEK293T cells.

[0124] Figure 3The base editing windows of APOBEC-nCas9-Ung and APOBEC-nCas9-Ung1 at 12 sites (6 C6 sites and 6 non-C6 sites) in HEK293T cells. a is the average base editing efficiency (C to G) of 20 pre-spacer sequences at 12 sites by the base editing systems APOBEC-nCas9-Ung and APOBEC-nCas9-Ung1; b is the C to G base editing efficiency of the base editing systems APOBEC-nCas9-Ung and APOBEC-nCas9-Ung1 at the C3-C8 positions of the 12 sites.

[0125] Figure 4 The results of base editing at C6 position of 17 sites in HEK293T cells by base editing systems APOBEC-nCas9-Ung and APOBEC-nCas9-Ung1. a is the C to G base editing efficiency at C6 position of 17 sites by base editing systems APOBEC-nCas9-Ung and APOBEC-nCas9-Ung1; b is the average base editing efficiency (C to A, C to T, C to G) of 18 pre-spacer sequences of 17 sites by base editing system APOBEC-nCas9-Ung; c is the average base editing efficiency (C to A, C to T, C to G) of 18 pre-spacer sequences of 17 sites by base editing system APOBEC-nCas9-Ung1.

[0126] Figure 5 The base editing windows of 12 sites (6 C6 sites and 6 non-C6 sites) of base editing systems APOBEC-nCas9-Ung1 and APOBEC(R33A)-nCas9-Rad51-Ung1 in HEK293T cells. a is the average base editing efficiency (C to G) of 20 pre-spacer sequences at 12 sites by base editing systems APOBEC-nCas9-Ung1 and APOBEC(R33A)-nCas9-Rad51-Ung1; b is the C to G base editing efficiency of base editing systems APOBEC-nCas9-Ung1 and APOBEC(R33A)-nCas9-Rad51-Ung1 at C3-C8 positions of 12 sites.

[0127] Figure 6The results of base editing at C6 position of 17 sites in HEK293T cells by base editing systems APOBEC-nCas9-Ung1 and APOBEC(R33A)-nCas9-Rad51-Ung1. a is the C to G base editing efficiency at C6 position of 17 sites by base editing systems APOBEC-nCas9-Ung1 and APOBEC(R33A)-nCas9-Rad51-Ung1; b is the average base editing efficiency (C to A, C to T, C to G) of 18 pre-spacer sequences at 17 sites by base editing system APOBEC(R33A)-nCas9-Rad51-Ung1.

[0128] Figure 7 The purity of the C to G base editing products at the C6 position of 17 sites in HEK293T cells using the base editing systems APOBEC-nCas9-Ung, APOBEC-nCas9-Ung1 and APOBEC(R33A)-nCas9-Rad51-Ung1. DETAILED DESCRIPTION

[0129] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0130] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0131] Example 1: Development and application of APOBEC-nCas9-Ung1 base editing system

[0132] In order to improve the C to G base editing efficiency of the GBE base editing system, the present invention developed a base editing system APOBEC-nCas9-Ung1, which includes cytosine deaminase APOBEC, nuclease nCas9 and uracil DNA glycosylase Ung1, as shown in the schematic diagram Figure 1 The following is a verification of the base editing efficiency of the base editing system APOBEC-nCas9-Ung1 at different gene sites in mammalian cells:

[0133] 1. Construction of base editor plasmid and gRNA plasmid

[0134] 1. Construction of base editor plasmid

[0135] The following recombinant expression vectors were artificially synthesized: APOBEC-nCas9-Ung recombinant expression vector, APOBEC-nCas9-cgUng recombinant expression vector and APOBEC-nCas9-Ung1 recombinant expression vector. Each vector is a circular plasmid.

[0136] APOBEC-nCas9-Ung recombinant expression vector (APOBEC-nCas9-Ung base editor plasmid): The nucleotide sequence of the vector is shown in sequence 6 in the sequence table, wherein positions 3977-4623 are the promoter sequence, positions 4695-5375 are the APOBEC coding gene sequence (cytosine deaminase APOBEC shown in coding sequence 1), positions 5430-9530 are the nCas9 coding gene sequence (nuclease nCas9 shown in coding sequence 2), positions 1-663 are the Ung coding gene sequence, and positions 770-946 are the terminator sequence.

[0137] APOBEC-nCas9-Ung1 recombinant expression vector (APOBEC-nCas9-Ung1 base editor plasmid): The nucleotide sequence of the APOBEC-nCas9-Ung1 recombinant expression vector is the sequence obtained by replacing positions 1-663 of the APOBEC-nCas9-Ung recombinant expression vector sequence with the coding gene sequence of Ung1 shown in sequence 7 (coding uracil DNA glycosylase Ung1 shown in sequence 3).

[0138] APOBEC-nCas9-cgUng recombinant expression vector (APOBEC-nCas9-cgUng base editor plasmid): The nucleotide sequence of the APOBEC-nCas9-cgUng recombinant expression vector is the sequence obtained by replacing positions 1-663 of the APOBEC-nCas9-Ung recombinant expression vector sequence with the coding gene sequence of cgUng shown in sequence 10.

[0139] 2. Construction of gRNA plasmid

[0140] The following recombinant expression vectors were artificially synthesized: RNF2-gRNA recombinant expression vector, VISTA hs267-gRNA recombinant expression vector, RP11-177B4-1-gRNA recombinant expression vector, RP1-97D16-gRNA recombinant expression vector, PSMB2-1-gRNA recombinant expression vector, RP11-177B4-3-gRNA recombinant expression vector, RP11-582C12-2-gRNA recombinant expression vector, HIRA-1-gRNA recombinant expression vector, PPP1R12C-site3-gRNA recombinant expression vector, RP11-399K21-2-gRNA recombinant expression vector, FANCY-gRNA recombinant expression vector, FANCF-site4-gRNA recombinant expression vector, DNMT3B-OTS1-gRNA recombinant expression vector, HIRA-2-gRNA recombinant expression vector. RNA recombinant expression vector, VEGFA-gRNA recombinant expression vector, EMX1-site7-gRNA recombinant expression vector, RP11-399K21-1-gRNA recombinant expression vector, PPP1R12C-site12-gRNA recombinant expression vector, HEK3-gRNA recombinant expression vector, HEK4-gRNA recombinant expression vector, PPP1R12C-site2-gRNA recombinant expression vector, PPP1R12C-site4-gRNA recombinant expression vector, PPP1R12C-site6-gRNA recombinant expression vector, EMX1-gRNA recombinant expression vector, EMX1-site4-gRNA recombinant expression vector, EMX1-site6-gRNA recombinant expression vector. Each vector is a circular plasmid.

[0141] RNF2-gRNA recombinant expression vector: The nucleotide sequence of the RNF2-gRNA recombinant expression vector is shown in sequence 8 in the sequence table, wherein positions 72-312 are the promoter sequence, positions 322-417 are the gRNA coding sequence (positions 322-341 are the pre-spacer series, positions 342-417 are the gRNA backbone sequence), and positions 1913-2034 are the terminator sequence.

[0142] The nucleotide sequences of other gRNA recombinant expression vectors are similar to that of the RNF2-gRNA recombinant expression vector. It is only necessary to replace the pre-spacer sequence shown at positions 322-341 in the nucleotide sequence of the RNF2-gRNA recombinant expression vector with the pre-spacer sequence corresponding to the corresponding target gene of other gRNA recombinant expression vectors.

[0143] Table 1. Pre-spacer sequences and amplification primers of gRNA plasmids

[0144]

[0145] Note: The first base site from the 5' end in each pre-spacer sequence is recorded as C1, the second base site is recorded as C2, and so on. The twentieth base site is recorded as C20.

[0146] 2. Base editing at different gene loci in mammalian cells

[0147] HEK293T cells (ATCC, CRL-3216), HepG2 cells (ATCC, HB-8065) ​​or HeLa cells (ATCC, CCL-2) were cultured at 5×10 5 24-well plates were plated, and when the cells in each well grew to 40%-60%, the three base editor plasmids APOBEC-nCas9-Ung, APOBEC-nCas9-cgUng and APOBEC-nCas9-Ung1 were respectively transfected with gRNA plasmids targeting different editing sites into HEK293T cells, HeLa cells or HepG2 cells using Lipofectamine 2000 (Life, Invitrogen, 11668019) reagent at a concentration of 600 ng base editor plasmid and 300 ng gRNA plasmid. Each plasmid combination was transfected in triplicate, and 5 μg / ml puromycin (Merck, USA) was added to the culture medium 24 hours after transfection. After 120 hours of transfection, genomic DNA was extracted using a rapid DNA extraction solution (Epicentre, USA), and the 200bp-300bp region near the edited site was PCR amplified using Taq DNA polymerase (Kangwei Century, China) (the amplification primer sequences are shown in Table 1), and the PCR products were subjected to high-throughput sequencing (Genwizhi, China), and the C to G base editing efficiency was calculated (C to G editing efficiency = C to G edited reads / total reads * 100%).

[0148] 3. Effects of uracil DNA glycosylases from different sources on the base editing efficiency of the GBE base editing system

[0149] In order to improve the performance of the GBE base editing system, the human uracil DNA glycosylase Ung in the GBE base editing system (base editing system APOBEC-nCas9-Ung) was replaced with uracil DNA glycosylase cgUng from Corynebacterium glutamicum and uracil DNA glycosylase Ung1 from Saccharomyces cerevisiae, respectively, to construct the base editing systems APOBEC-nCas9-cgUng and APOBEC-nCas9-Ung1, respectively. The base editing systems with different sources of uracil DNA glycosylase (APOBEC-nCas9-Ung, APOBEC-nCas9-cgUng, APOBEC-nCas9-Ung1) were used to perform base editing at five sites (HIRA-2, HEK3, EMX1-site6, PPP1R12C-site12, FANCY-site4) in HEK293T cells, and the base editing efficiency from C to G was calculated.

[0150] The results are as follows Figure 2 As shown. The results showed that compared with the base editing system APOBEC-nCas9-Ung, the C to G base editing efficiency of the base editing system APOBEC-nCas9-cgUng was significantly reduced at 2 of the 5 sites (HIRA-2, HEK3), the C to G base editing efficiency of 2 sites (EMX1-site6, FANCY-site4) was significantly improved, and there was no significant difference in 1 site (PPP1R12C-site12). In contrast, the C to G base editing efficiency of the base editing system APOBEC-nCas9-Ung1 was significantly higher than that of APOBEC-nCas9-Ung at 4 of the 5 sites. Therefore, the base editing system APOBEC-nCas9-Ung1 was selected as the research object, and further statistics and analysis of the base editing window and base editing efficiency were performed based on more sites.

[0151] 4. Statistics and analysis of base editing window and base editing efficiency

[0152] 1. Statistics and analysis of base editing windows

[0153] In order to explore the base editing window of the base editing system APOBEC-nCas9-Ung1, 12 sites (6 C6 sites and 6 non-C6 sites. The 6 C6 sites are as follows: EMX1, HIRA-2, FANCY, EMX1-site4, EMX1-site6, RP11-177B4-3. The 6 non-C6 sites are as follows: PPP1R12C-site12, HEK3, HEK4, PPP1R12C-site2, PPP1R12C-site4, PPP1R12C-site6.) were selected for base editing in HEK293T cells.

[0154] The base editing efficiency of APOBEC-nCas9-Ung and APOBEC-nCas9-Ung1 at 12 sites from C to G is shown in Figure 2. Figure 3 The results showed that both base editing systems (APOBEC-nCas9-Ung and APOBEC-nCas9-Ung1) could perform a small amount of C to G editing at the C5 and C7 positions of the pre-spacer sequence. Similar to the base editing system APOBEC-nCas9-Ung, the base editing system APOBEC-nCas9-Ung1 mainly edited the C6 position of the pre-spacer sequence.

[0155] 2. Statistics and analysis of base editing efficiency

[0156] The APOBEC-nCas9-Ung and APOBEC-nCas9-Ung1 base editing systems were used to modify 17 different C6 sites in HEK293T cells (VISTA hs267, RP11-177B4-1, RNF2, RP1-97D16, PSMB2-1, RP11-177B4-3, RP11-582C12-2, HIRA-1, PPP1R12C-site3, RP11-399K21-2, FANCY, FANCF-site4, DNMT3B-OTS1, HIRA-2, VEGFA, EMX1-site e7, RP11-399K21-1) for base editing, analyze and calculate the average base editing efficiency of 18 pre-spacer sequences of APOBEC-nCas9-Ung and APOBEC-nCas9-Ung1 in 17 sites, including C2, C3, C4, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19 and C20. Taking C2 as an example, the average base editing efficiency of C2 = the sum of the editing efficiencies of the C2 sites / the number of gRNAs containing the C2 sites * 100%. If a C2 site is a non-C base, the editing efficiency of the C2 site is 0.

[0157] The results are as follows Figure 4 and Table 2. The results showed that the base editing systems APOBEC-nCas9-Ung and APOBEC-nCas9-Ung1 mainly edited the C6 position on the protoplast sequence, indicating that the change in the Ung component did not affect the editing window. However, as Ung was changed to Ung1, the average C to G base editing efficiency at the C6 position increased from 15.54% to 23.48%, an increase of 51.14%. Specifically, compared with the base editing system APOBEC-nCas9-Ung, the base editing system APOBEC-nCas9-Ung1 significantly improved the C to G base editing efficiency at the C6 position on 12 sites (RP11-177B4-1, PSMB2-1, RP1-97D16, HIRA-1, RP11-177B4-3, RP11-399K21-2, RP11-582C12-2, FANCF-site4, EMX1-site7, DNMT3B-OTS1, PPP1R12C-site3, RP11-399K21-1). Among 17 different sites, the base editing efficiency of the base editing system APOBEC-nCas9-Ung1 at some sites (RP11-177B4-1, PSMB2-1, HIRA-1, RP11-177B4-3) was 2-3 times that of the base editing system APOBEC-nCas9-Ung. For example, the C to G base editing efficiency at the RP11-177B4-1 site increased from 26.80% to 52.30%, the C to G base editing efficiency at the PSMB2-1 site increased from 25.73% to 43.90%, the C to G base editing efficiency at the RP11-177B4-3 site increased from 14.00% to 24.90%, and the C to G base editing efficiency at the HIRA-1 site increased from 10.00% to 29.90%. In addition, the C to G base editing efficiency at the three sites of FANCF-site4, EMX1-site7 and DNMT3B-OTS1 was increased from less than 4% to nearly 20%.

[0158] Table 2. Base editing efficiency of C to G in HEK293T cells using base editing systems APOBEC-nCas9-Ung, APOBEC-nCas9-Ung1 and APOBEC(R33A)-nCas9-Rad51-Ung1 (C6 position)

[0159]

[0160] From the above sites, 7 different sites (DNMT3B-OTS1, HIRA-1, PPP1R12C-site3, FANCF-site4, RP11-582C12-2, RNF2, RP11-177B4-3) were selected and base editing was performed in HepG2 and HeLa cells using the base editing systems APOBEC-nCas9-Ung and APOBEC-nCas9-Ung1, respectively.

[0161] The results are shown in Tables 3 and 4, which show that in HepG2 cells, the C to G base editing efficiency of APOBEC-nCas9-Ung1 at the C6 position on three sites (DNMT3B-OTS1, HIRA-1, FANCF-site4) was significantly improved compared with the base editing system APOBEC-nCas9-Ung. In HeLa cells, the C to G base editing efficiency of APOBEC-nCas9-Ung1 at the C6 position on six sites (DNMT3B-OTS1, HIRA-1, PPP1R12C-site3, FANCF-site4, RP11-582C12-2, and RNF2) was significantly improved compared with the base editing system APOBEC-nCas9-Ung.

[0162] Table 3. Base editing efficiency of C to G in HepG2 cells using base editing systems APOBEC-nCas9-Ung, APOBEC-nCas9-Ung1 and APOBEC(R33A)-nCas9-Rad51-Ung1 (C6 position)

[0163]

[0164] Table 4. Base editing efficiency of C to G in HeLa cells by base editing systems APOBEC-nCas9-Ung, APOBEC-nCas9-Ung1 and APOBEC(R33A)-nCas9-Rad51-Ung1 (C6 position)

[0165]

[0166] The above experimental results show that in mammalian cells, different editing sites are base edited using the base editing system APOBEC-nCas9-Ung1, and its C to G base editing efficiency is improved compared with the base editing system APOBEC-nCas9-Ung.

[0167] Example 2: Development of the APOBEC(R33A)-nCas9-Rad51-Ung1 base editing system

[0168] In order to further improve the efficiency of C to G base editing and the purity of base editing products, the present invention developed a base editing system APOBEC (R33A) -nCas9-Rad51-Ung1, which includes cytosine deaminase APOBEC (R33A), nuclease nCas9, single-stranded DNA binding protein Rad51 and uracil DNA glycosylase Ung1, as shown in the schematic diagram Figure 1 The following is a verification of the base editing efficiency of the base editing system APOBEC (R33A) -nCas9-Rad51-Ung1 at different gene sites in mammalian cells:

[0169] 1. Construction of base editor plasmid and gRNA plasmid

[0170] 1. Construction of base editor plasmid

[0171] The following recombinant expression vectors were artificially synthesized: APOBEC-nCas9-Ung recombinant expression vector, APOBEC-nCas9-Ung1 recombinant expression vector and APOBEC(R33A)-nCas9-Rad51-Ung1 recombinant expression vector. Each vector is a circular plasmid.

[0172] APOBEC-nCas9-Ung recombinant expression vector (APOBEC-nCas9-Ung base editor plasmid): same as Example 1.

[0173] APOBEC-nCas9-Ung1 recombinant expression vector (APOBEC-nCas9-Ung1 base editor plasmid): same as Example 1.

[0174] APOBEC(R33A)-nCas9-Rad51-Ung1 recombinant expression vector (APOBEC(R33A)-nCas9-Rad51-Ung1 base editor plasmid): The nucleotide sequence of the vector is shown in sequence 9 in the sequence list, wherein positions 4391-5037 are the promoter sequence, positions 5109-5789 are the coding gene sequence of APOBEC(R33A) (coding cytosine deaminase APOBEC(R33A) shown in sequence 4), positions 5844-9944 are the coding gene sequence of nCas9, positions 10089-10430 are the coding gene sequence of Rad51 (single-stranded DNA binding protein Rad51 shown in coding sequence 5), positions 1-1077 are the coding gene sequence of Ung1, and positions 1184-1360 are the terminator sequence.

[0175] 2. Construction of gRNA plasmid

[0176] gRNA recombinant expression vector: same as Example 1.

[0177] 2. Base editing at different sites in mammalian cells

[0178] HEK293T cells (ATCC, CRL-3216), HepG2 cells (ATCC, HB-8065) ​​or HeLa cells (ATCC, CCL-2) were cultured at 5×10 5 24-well plates were plated, and when the cells in each well grew to 40%-60%, the three base editor plasmids APOBEC-nCas9-Ung, APOBEC-nCas9-Ung1 and APOBEC(R33A)-nCas9-Rad51-Ung1 were respectively transfected with gRNA plasmids targeting different editing sites into HEK293T cells, HeLa cells or HepG2 cells using Lipofectamine 2000 (Life, Invitrogen, 11668019) reagent according to the amount of 600 ng base editor plasmid and 300 ng gRNA plasmid. Each plasmid combination was transfected in triplicate, and 5 μg / ml puromycin (Merck, USA) was added to the culture medium 24 hours after transfection. 120 hours after transfection, genomic DNA was extracted using a rapid DNA extraction solution (Epicentre, USA), and the 200bp-300bp region near the edited site was PCR amplified using Taq DNA polymerase (Kangwei Century, China) (the amplification primer sequences are shown in Table 1), and the PCR products were subjected to high-throughput sequencing (Genwizhi, China), and the base editing efficiency of C to G and the purity of the base editing products were calculated (purity of base editing products from C to G = base editing efficiency from C to G / (base editing efficiency from C to G + base editing efficiency from C to A + base editing efficiency from C to T)*100%).

[0179] III. Statistics and analysis of base editing window, base editing efficiency and product purity

[0180] 1. Statistics and analysis of base editing windows

[0181] To explore the base editing window of the base editing system APOBEC(R33A)-nCas9-Rad51-Ung1, 12 sites (6 C6 sites and 6 non-C6 sites. The 6 C6 sites are as follows: EMX1, HIRA-2, FANCY, EMX1-site4, EMX1-site6, RP11-177B4-3. The 6 non-C6 sites are as follows: PPP1R12C-site12, HEK3, HEK4, PPP1R12C-site2, PPP1R12C-site4, PPP1R12C-site6) were selected for base editing in HEK293T cells.

[0182] The base editing efficiency of APOBEC-nCas9-Ung1 and APOBEC(R33A)-nCas9-Rad51-Ung1 at 12 sites from C to G is shown in Figure 2. Figure 5 As shown. The results showed that the base editing system APOBEC-nCas9-Ung1 can perform a small amount of C to G editing at the C5 and C7 positions of the pre-spacer sequence, while the base editing system APOBEC(R33A)-nCas9-Rad51-Ung1 can perform a small amount of C to G editing at the C8 position in addition to the C5 and C7 positions. Similar to the base editing system APOBEC-nCas9-Ung1, the base editing system APOBEC(R33A)-nCas9-Rad51-Ung1 also mainly edits the C6 position of the pre-spacer sequence.

[0183] 2. Statistics and analysis of base editing efficiency

[0184] The APOBEC-nCas9-Ung1 and APOBEC(R33A)-nCas9-Rad51-Ung1 base editing systems were used to target 17 different C6 sites in HEK293T cells (VISTA hs267, RP11-177B4-1, RNF2, RP1-97D16, PSMB2-1, RP11-177B4-3, RP11-582C12-2, HIRA-1, PPP1R12C-site3, RP11-399K21-2, FANCY, FANCF-site4, DNMT3B-OTS1, HIRA-2, VEGFA, EMX1-site7, RP11-3 99K21-1) base editing was performed, and the average base editing efficiency of 18 pre-spacer sequences of the base editing systems APOBEC-nCas9-Ung1 and APOBEC (R33A)-nCas9-Rad51-Ung1 was analyzed and calculated in 17 sites, including C2, C3, C4, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19 and C20. Taking C2 as an example, the average base editing efficiency of C2 = the sum of the editing efficiencies of the C2 sites / the number of gRNAs containing the C2 sites * 100%. If a C2 site is a non-C base, the editing efficiency of the C2 site is 0.

[0185] The results are as follows Figure 6As shown. The results showed that the base editing system APOBEC (R33A) -nCas9-Rad51-Ung1 mainly achieved C to G base editing at the C6 position of the pre-spacer sequence, and its C to G base editing efficiency was 8.70%-72.10%, while the C to G base editing efficiency of the base editing system APOBEC-nCas9-Ung1 was 2.63%-52.3%. The average editing efficiency of the base editing system APOBEC (R33A) -nCas9-Rad51-Ung1 at 17 sites was 30.88%, which was 31.50% and 98.75% higher than that of the base editing system APOBEC-nCas9-Ung1 and the base editing system APOBEC-nCas9-Ung, respectively. Specifically, the C to G base editing efficiency of the base editing system APOBEC (R33A) -nCas9-Rad51-Ung1 at 10 sites (VISTA hs267, RNF2, RP1-97D16, RP11-177B4-3, RP11-582C12-2, HIRA-1, PPP1R12C-site3, FANCE, HIRA-2, VEGFA) was significantly higher than that of the base editing system APOBEC-nCas9-Ung1. Among them, the C to G base editing efficiency of 5 sites was about 1.5 times that of the base editing system APOBEC-nCas9-Ung1 (VISTA The C to G base editing efficiency of hs267 site increased from 47.23% to 72.10%; the C to G base editing efficiency of RP1-97D16 site increased from 33.07% to 50.07%; the C to G base editing efficiency of RP11-177B4-3 site increased from 24.90% to 39.43%; the C to G base editing efficiency of RP11-582C12-2 site increased from 22.87% to 33.07%; the C to G base editing efficiency of PPP1R12C-site3 site increased from 1 The C to G base editing efficiency of the APOBEC-nCas9-Ung1 system was increased from 4.00% to 23.53%), one site was about twice that of the APOBEC-nCas9-Ung1 system (the C to G base editing efficiency of the RNF2 site increased from 25.40% to 50.23%), and two sites were about three times that of the APOBEC-nCas9-Ung1 system (the C to G base editing efficiency of the HIRA-2 site increased from 5.53% to 16.27%; the C to G base editing efficiency of the VEGFA site increased from 6.07% to 16.17%). In addition, the C to G base editing efficiency of the APOBEC(R33A)-nCas9-Rad51-Ung1 system at the FANCY site reached 22.90%, while the C to G base editing efficiency of the APOBEC-nCas9-Ung1 system at this site was only 2.63%.

[0186] From the above sites, 7 different sites (DNMT3B-OTS1, HIRA-1, PPP1R12C-site3, FANCF-site4, RP11-582C12-2, RNF2, RP11-177B4-3) were selected for base editing experiments in HepG2 and HeLa cells.

[0187] The results are shown in Tables 3 and 4. The results showed that in HepG2 cells and HeLa cells, compared with the base editing system APOBEC-nCas9-Ung1, the C to G base editing efficiency of APOBEC(R33A)-nCas9-Rad51-Ung1 at the C6 position of 7 sites was significantly improved.

[0188] The above experimental results show that in mammalian cells, different editing sites are base edited using the base editing system APOBEC(R33A)-nCas9-Rad51-Ung1, and its C to G base editing efficiency is improved compared with the base editing systems APOBEC-nCas9-Ung1 and APOBEC-nCas9-Ung.

[0189] 3. Statistics and analysis of base editing product purity

[0190] Three base editing systems, APOBEC-nCas9-Ung, APOBEC-nCas9-Ung1 and APOBEC(R33A)-nCas9-Rad51-Ung1, were used to modify 17 different C6 sites in HEK293T cells (VISTA hs267, RP11-177B4-1, RNF2, RP1-97D16, PSMB2-1, RP11-177B4-3, RP11-582C12-2, HIRA-1, PPP1R12C-site3, RP11-399K21-2, FANCY, FANCF-site4, DNMT3B-OTS1, HIRA-2, VEGFA, EMX1-site7, RP11-399K21-1) were base edited, and the purity of the base editing products from C to G at the C6 position of 17 sites in HEK293T cells by the base editing systems APOBEC-nCas9-Ung, APOBEC-nCas9-Ung1 and APOBEC(R33A)-nCas9-Rad51-Ung1 were analyzed and calculated.

[0191] The results are as follows Figure 7The results showed that the purities of the C to G base editing products of the three base editing systems APOBEC-nCas9-Ung, APOBEC-nCas9-Ung1 and APOBEC(R33A)-nCas9-Rad51-Ung1 were 16.41%-93.64%, 16.04%-90.77% and 35.57%-93.92%, respectively. The base editing system APOBEC-nCas9-Ung has base editing products with a purity of more than 80% at 6 sites (RP11-177B4-1, VISTA hs267, PSMB2-1, RP1-97D16, HIRA-1, FANCF-site4), 60%-80% at 5 sites (RNF2, RP11-177B4-3, RP11-399K21-2, RP11-582C12-2, EMX1-site7), and less than 60% at 6 sites (DNMT3B-OTS1, PPP12C-site3, RP11-399K21-1-1, VEGFA, HIRA-2, FANCE). The base editing system APOBEC-nCas9-Ung1 has base editing products with a purity of more than 80% at 5 sites (RP11-177B4-1, VISTA hs267, PSMB2-1, RP1-97D16, and HIRA-1), 60%-80% at 4 sites (RP11-177B4-3, RP11-399K21-2, FANCF-site4, and EMX1-site7), and less than 60% at 8 sites (RNF2, RP11-582C12-2, DNMT3B-OTS1, PPP12C-site3, RP11-399K21-1, VEGFA, HIRA-2, and FANCE). The base editing system APOBEC (R33A)-nCas9-Rad51-Ung1 has 8 sites (RP11-177B4-1, VISTAhs267, PSMB2-1, RP1-97D16, HIRA-1, RNF2, RP11-399K21-2, FANCF-site4) with a purity of base editing products exceeding 80%, 6 sites (RP11-177B4-3, RP11-582C12-2, EMX1-site7, DNMT3B-OTS1, HIRA-2, FANCY) with a purity between 60% and 80%, and 3 sites (PPP1R12C-site3, RP11-399K21-1-1, VEGFA) with a purity of base editing products less than 60%.The average base editing product purities of the base editing systems APOBEC-nCas9-Ung, APOBEC-nCas9-Ung1 and APOBEC(R33A)-nCas9-Rad51-Ung1 were 64.25%, 60.33% and 73.44%, respectively.

[0192] The above experimental results show that in mammalian cells, the base editing system APOBEC(R33A)-nCas9-Rad51-Ung1 achieved higher C to G conversion purity compared with the base editing systems APOBEC-nCas9-Ung and APOBEC-nCas9-Ung1.

[0193] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims. Sequence Listing <110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences <120> A recombinant glycosylase base editing system and its application <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 227 <212> PRT <213> Artificial Sequence <400> 1 Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg Arg 1 5 10 15 Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu Arg 20 25 30 Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly Arg His Ser 35 40 45 Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His Val Glu Val Asn 50 55 60 Phe Ile Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro Asn Thr Arg 65 70 75 80 Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys Ser 85 90 95 Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val Thr Leu Phe 100 105 110 Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg Asn Arg Gln 115 120 125 Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln Ile Met Thr 130 135 140 Glu Gln Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn Tyr Ser Pro 145 150 155 160 Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp Val Arg Leu 165 170 175 Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys Leu 180 185 190 Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile Ala 195 200 205 Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp Ala 210 215 220 Thr Gly Leu 225 <210> 2 <211> 1367 <212> PRT <213> Artificial Sequence <400> 2 Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly 1 5 10 15 Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys 20 25 30 Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly 35 40 45 Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys 50 55 60 Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr 65 70 75 80 Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe 85 90 95 Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His 100 105 110 Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His 115 120 125 Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser 130 135 140 Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met 145 150 155 160 Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp 165 170 175 Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn 180 185 190 Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys 195 200 205 Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu 210 215 220 Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu 225 230 235 240 Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp 245 250 255 Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp 260 265 270 Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu 275 280 285 Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile 290 295 300 Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met 305 310 315 320 Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala 325 330 335 Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp 340 345 350 Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln 355 360 365 Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly 370 375 380 Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys 385 390 395 400 Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly 405 410 415 Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu 420 425 430 Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro 435 440 445 Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met 450 455 460 Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val 465 470 475 480 Val Asp Lys Gly Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn 485,490,495 Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu 500 505 510 Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr 515,520,525 Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys 530 535 540 Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val 545 550 555 560 Lys Gln Leu Lys Glu Asp Tyr Phe Lys Ile Glu Cys Phe Asp Ser 565 570 575 Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr 580 585 590 Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn 595 600 605 Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu 610 615 620 Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His 625 630 635 640 Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr 645 650 655 Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys 660 665 670 Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala 675 680 685 Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys 690 695 700 Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His 705 710 715 720 Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile 725 730 735 Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg 740 745 750 His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr 755 760 765 Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu 770 775 780 Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val 785 790 795 800 Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln 805 810 815 Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu 820 825 830 Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp 835 840 845 Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly 850 855 860 Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn 865 870 875 880 Tyr Trp Arg Gln Leu Asn Ala Lys Leu And Thr Gln Arg Lys Phe 885,890,895 Asp Asn With Thr Lys Ala Glu Arg Gly Gly Ser Glu With Asp Lys 900 905 910 Only Gly Phe With Lys Arg Gln Leu Val Glu Thr Arg Gln With Thr Lys 915,920,925 His Gln Ile Leu Asp Ser Arg With Asn Thr Lys Tyr Asp Glu 930,935,940 Asn Asp Lys With Arg Glu Val Val Lys With Thr Lys Ser Ser Lys 945 950 955 960 Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu 965,970,975 Ile Asn Asn Tyr His Ala His Asp Ala Tyr Leu Asn Ala Val Val 980,985,990 Gly Thr Ala Leu Ile Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val 995 1000 1005 Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys 1010 1015 1020 Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr 1025 1030 1035 Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn 1040 1045 1050 Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr 1055 1060 1065 Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg 1070 1075 1080 Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu 1085 1090 1095 Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg 1100 1105 1110 Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys 1115 1120 1125 Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu 1130 1135 1140 Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser 1145 1150 1155 Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe 1160 1165 1170 Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu 1175 1180 1185 Val Lys Lys Asp Leu Ile Ile Lys Pro Lys Lys Ser Leu Phe 1190 1195 1200 Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu 1205 1210 1215 Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn 1220 1225 1230 Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro 1235 1240 1245 Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His 1250 1255 1260 Tyr Leu Asp Glu Ile Ile Glu Gln Ile Glu Phe Serve Lys Arg 1265 1270 1275 There Is A Lion On Asp Only Asn On The Asp Lys On The Lion On Tyr 1280 1285 1290 Asn Lys His Arg Asp Lys Pro With Arg Glu Gln Ala Glu Asn Ile 1295 1300 1305 Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe 1310 1315 1320 Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr 1325 1330 1335 Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly 1340 1345 1350 Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 <210> 3 <211> 359 <212> PRT <213> Artificial Sequence <400> 3 Met Trp Cys Met Arg Arg Leu Pro Thr Asn Ser Val Met Thr Val Ala 1 5 10 15 Arg Lys Arg Lys Gln Thr Thr Ile Glu Asp Phe Phe Gly Thr Lys Lys 20 25 30 Ser Thr Asn Glu Ala Pro Asn Lys Lys Gly Lys Ser Gly Ala Thr Phe 35 40 45 Met Thr Ile Thr Asn Gly Ala Ala Ile Lys Thr Glu Thr Lys Ala Val 50 55 60 Ala Lys Glu Ala Asn Thr Asp Lys Tyr Pro Ala Asn Ser Asn Ala Lys 65 70 75 80 Asp Val Tyr Ser Lys Asn Leu Ser Ser Asn Leu Arg Thr Leu Leu Ser 85 90 95 Leu Glu Leu Glu Thr Ile Asp Asp Ser Trp Phe Pro His Leu Met Asp 100 105 110 Glu Phe Lys Lys Pro Tyr Phe Val Lys Leu Lys Gln Phe Val Thr Lys 115 120 125 Glu Gln Ala Asp His Thr Val Phe Pro Pro Ala Lys Asp Ile Tyr Ser 130 135 140 Trp Thr Arg Leu Thr Pro Phe Asn Lys Val Lys Val Val Ile Ile Gly 145 150 155 160 Gln Asp Pro Tyr His Asn Phe Asn Gln Ala His Gly Leu Ala Phe Ser 165 170 175 Val Lys Pro Pro Thr Pro Ala Pro Pro Ser Leu Lys Asn Ile Tyr Lys 180 185 190 Glu Leu Lys Gln Glu Tyr Pro Asp Phe Val Glu Asp Asn Lys Val Gly 195 200 205 Asp Leu Thr His Trp Ala Ser Gln Gly Val Leu Leu Leu Asn Thr Ser 210 215 220 Leu Thr Val Arg Ala His Asn Ala Asn Ser His Ser Lys His Gly Trp 225 230 235 240 Glu Thr Phe Thr Lys Arg Val Val Gln Leu Leu Ile Gln Asp Arg Glu 245 250 255 Ala Asp Gly Lys Ser Leu Val Phe Leu Leu Trp Gly Asn Asn Ala Ile 260 265 270 Lys Leu Val Glu Ser Leu Leu Gly Ser Thr Ser Val Gly Ser Gly Ser 275 280 285 Lys Tyr Pro Asn Ile Met Val Met Lys Ser Val His Pro Ser Pro Leu 290 295 300 Ser Ala Ser Arg Gly Phe Phe Gly Thr Asn His Phe Lys Met Ile Asn 305 310 315 320 Asp Trp Leu Tyr Asn Thr Arg Gly Glu Lys Met Ile Asp Trp Ser Val 325 330 335 Val Pro Gly Thr Ser Leu Arg Glu Val Gln Glu Ala Asn Ala Arg Leu 340 345 350 Glu Ser Glu Ser Lys Asp Pro 355 <210> 4 <211> 227 <212> PRT <213> Artificial Sequence <400> 4 Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg Arg 1 5 10 15 Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu Ala 20 25 30 Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly Arg His Ser 35 40 45 Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His Val Glu Val Asn 50 55 60 Phe Ile Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro Asn Thr Arg 65 70 75 80 Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys Ser 85 90 95 Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val Thr Leu Phe 100 105 110 Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg Asn Arg Gln 115 120 125 Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln Ile Met Thr 130 135 140 Glu Gln Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn Tyr Ser Pro 145 150 155 160 Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp Val Arg Leu 165 170 175 Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys Leu 180 185 190 Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile Ala 195 200 205 Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp Ala 210 215 220 Thr Gly Leu 225 <210> 5 <211> 114 <212> PRT <213> Artificial Sequence <400> 5 Met Ala Met Gln Met Gln Leu Glu Ala Asn Ala Asp Thr Ser Val Glu 1 5 10 15 Glu Glu Ser Phe Gly Pro Gln Pro Ile Ser Arg Leu Glu Gln Cys Gly 20 25 30 Ile Asn Ala Asn Asp Val Lys Lys Leu Glu Glu Ala Gly Phe His Thr 35 40 45 Val Glu Ala Val Ala Tyr Ala Pro Lys Lys Glu Leu Ile Asn Ile Lys 50 55 60 Gly Ile Ser Glu Ala Lys Ala Asp Lys Ile Leu Ala Glu Ala Ala Lys 65 70 75 80 Leu Val Pro Met Gly Phe Thr Thr Ala Thr Glu Phe His Gln Arg Arg 85 90 95 Ser Glu Ile Ile Gln Ile Thr Thr Gly Ser Lys Glu Leu Asp Lys Leu 100 105 110 Leu Gln <210> 6 <211> 9599 <212> DNA <213> Artificial Sequence <400> 6 atgtttggag agagctggaa gaagcacctc agcggggagt tcgggaaacc gtattttatc 60 aagctaatgg gatttgttgc agaagaaaga aagcattaca ctgttatcc acccccacac 120 caagtcttca cctggaccca gatgtgtgac ataaaagatg tgaaggttgt catcctggga 180 caggatccat atcatggacc taatcaagct cacgggctct gctttagtgt tcaaaggcct 240 gttccgcctc cgcccagttt ggagaacatt tataaagagt tgtctacaga catagaggat 300 tttgttcatc ctggccatgg agatttatct gggtgggcca agcaaggtgt tctccttctc 360 aacgctgtcc tgcccatcaa gccaactctc ataaggagcg aggctgggag 420 cagttcactg atgcagttgt gtcctggcta aatcagaact cgaatggcct tgttttcttg 480 ctctggggct cttatgctca gaagaagggc agtgccattg ataggaagcg gcaccatgta 540 ctacagacgg ctcatccctc cccttgtca gtgtatagag ggttctttgg atgtagacac 600 tttcaaga ccaatgagct gctgcagaag tctggcaga agcccattga ctggaggag 660 ctgtcgggggg ggagcccaa gagaagcgg aaggtgtagt gtacaagtaa cccgggaatt 720 cggtacgctg aaatcaccag tctctcta caaatctatc tctcttatt ttctccataa 780 ataatgtgtg agtagtttcc cgataaggga aattaggtt cttataggtt ttcgctcatg 840 tgttgagcat ataagaaacc cttagtatgt atttgtattt gtaaaatact tctatcaata 900 aaatttctaa ttcctaaaac caaatccag tactaaaatc cagatctcct aaagtcccta 960 tagatcttg tcgtgaat aaaccagaca cgagacgact aaacctggag cccagacgcc 1020 gttcgaagct agaagtaccg cttaggcagg aggccgttag ggaaagatg ctaggcagg 1080 gttggttacg ttgactcccc cgtaggttg gtttaaatat gatgaagtgg acggaaggaa 1140 ggaggagac aagggat aaggttgcag gccctgtgca agtaagaag atggaattt 1200 gatagaggta cgctactata cttatactat acgctaaggg aatgctgta ttataccct 1260 atacccccta ataacccctt atcaatttaa gaataatcc gcataaggccc ccgcttaaa 1320 attggtatca gagccatgaa taggtctatg accaaaactc aagaggattaa aacctcacca 1380 aaatacgaaa gagttcttaa ctctaaagat aaaagatctt tcaagatcaa aactagttcc 1440 ctcacaccgg tgacggggat cgcatgcgat atctcgagat ctagcttggc gtaatcatgg 1500 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc 1560 ggaagcataa agtgttaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg 1620 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc 1680 ggccaacgcg cggggagagg cggtttgcgt attgggcggc cttccgcttc ctcgctcact 1740 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 1800 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 1860 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 1920 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 1980 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 2040 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 2100 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 2160 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 2220 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 2280 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 2340 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 2400 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 2460 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 2520 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 2580 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 2640 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc 2700 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg 2760 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct 2820 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca 2880 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg 2940 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg 3000 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc 3060 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag 3120 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg 3180 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag 3240 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat 3300 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg 3360 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca 3420 gcatcttta ctttcaccag cgttctctggg tgagcaaaa caggaaggca aaatgccgca 3480 aaaaagggaa taagggcgac acggaatgt tgaatactca tactctcct tttcaat 3540 tattgaagca tttatcaggg ttattgtctc atgagcggat acatattga atgtatttag 3600 aaaaataaac aaataggggt tccgcgcaca ttccccgaa aagtgccacc tgccagtgcc 3660 aagctaattc gagctcggta cctgacccgg tcgtgcccct cttagagat aatgagcatt 3720 gcatgtctaa gttataaaaa attaccacat attttttg tcacacttgt ttgaagtgca 3780 gtttatctat ctttatacat atatttaac tttacttac gaataatata atctatagta 3840 ctacaataat atcagtgtttt tagagaatca tataaatgaa cagttagaca tggtctaaag 3900 gatatgca tgaagaatct gcttaggtt aggcgtttg cgctgcttcg cgatgtacgg 3960 gccagatata cgcgttgaca ttgattattg actagttatt atagtaatc aattacgggg 4020 tcattagttc atagcccata tatggattc cgcgttacat aacttacggt aaatggcccg 4080 cctggctgac cgcccaacga cccccgccca tgacgtca taatgacgta tgttcccata 4140 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc 4200 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac 4260 ggtaaatggc ccgcctggca tttgcccag tacatgacct tatgggactt tcctacttgg 4320 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc 4380 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc 4440 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc 4500 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct 4560 ctctggctaa ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat 4620 agggagaccc aagctggcta gcgtttaaac ttaagcttgt cgacggatcc atgccaaaga 4680 agaagaggaa ggtttcatcg gagaccggcc ctgttgctgt tgaccccacc ctgcggcgga 4740 gaatcgagcc acacgagttc gaggtgttct tcgacccaag ggagctccgc aaggagacgt 4800 gcctcctgta cgagatcaac tggggcggca ggcactccat ctggaggcac accagccaaa 4860 acaccaacaa gcacgtggag gtcaacttca tcgagaagtt caccaccgag aggtacttct 4920 gcccaaacac ccgctgctcc atcacctggt tcctgtcctg gagcccatgc ggcgagtgct 4980 ccagggccat caccgagttc ctcagccgct acccacacgt caccctgttc atctacatcg 5040 ccaggctcta ccaccacgcc gacccaagga acaggcaggg cctccgcgac ctgatctcca 5100 gcggcgtgac catccaaatc atgaccgagc aggagtccgg ctactgctgg aggaacttcg 5160 tcaactactc cccaagcaac gaggcccact ggccaaggta cccacacctc tgggtgcgcc 5220 tctacgtgct cgagctgtac tgcatcatcc tcggcctgcc accatgcctc aacatcctga 5280 ggcgcaagca accacagctg accttcttca ccatcgccct ccaaagctgc cactaccaga 5340 ggctcccacc acacatcctg tgggctaccg gcctcaagtc cggcagcgag acgccaggca 5400 cctccgagag cgctacgcct gaacttaagg acaagaagta ctcgatcggc ctcgccatcg 5460 ggacgaactc agttggctgg gccgtgatca ccgacgagta caaggtgccc tctaagaagt 5520 tcaaggtcct ggggaacacc gaccgccatt ccatcaagaa gaacctcatc ggcgctctcc 5580 tgttcgacag cggggagacc gctgaggcta cgaggctcaa gagaaccgct aggcgccggt 5640 acacgagaag gaaacagg atctgctacc tccaagagat tttctccaac gagatggcca 5700 5760 agcacgagcg gcatcccatc ttcggcaaca tcgtggacga ggttgcctac cacgagaagt 5820 accctacgat ctaccatctg cggaagaagc tcgtggactc caccgataag gcggacctca 5880 gactgatcta cctcgctctg gcccacatga tcaagttccg cggccatttc ctgatcgagg 5940 gggatctcaa cccagacaac agcgatgttg acaagctgtt catccaactc gtgcagacct 6000 aaaccaact cttcgaggag aacccgatca acgcctctgg cgtggacgcg aaggctatcc 6060 tgtccgcgag gctctcgaag tccaggaggc tggagaacct gatcgctcag ctcccaggcg 6120 yours cggcctgttc gggaacctca tcgctctcag cctggggctc accccgaact 6180 tcaagtcgaa cttcgatctc gctgaggacg ccaagctgca actctccaag gacacctacg 6240 acgatgacct cgataacctc ctggcccaga tcggcgatca atacgcggac ctgttcctcg 6300 ctgccaagaa cctgtcggac gccatcctcc tgtcagatat cctccgcgtg aacaccgaga 6360 tcacgaaggc tccactctct gcctccatga tcaagcgcta cgacgagcac catcaggatc 6420 tgaccctcct gaaggcgctg gtccgccaac agctccccgga gaagtcaag gagattttct 6480 tcgatcagtc gaagacggc tacgctgggt acatcgacgg cggggcctca caagaggagt 6540 tctacaagtt catcaagcca atcctggaga agatggacgg cacgggagg ctcctggtga 6600 agctcaacag ggaggacctc ctgcggaagc agaaacctt cgataacggc agcatccccc 6660 accaaatcca tctcggggag ctgcacgcca tcctgagaag gcaagaggac ttctaccctt 6720 tcctcaagga taaccgggag aagatcgaga agatcctgac cttcagaatc ccatactacg 6780 tcggccctct cgcgcgggg aactcaagat tcgcttggat gacccgcaag tctgaggaga 6840 ccatcacgcc gtggaacttc gaggaggtgg tggacaaggg cgctagcgct cagtcgttca 6900 tcgagaggat gaccaacttc gaagaaacc tgcccaacga gaaggtgctc cctaagcact 6960 cgctcctgta cgagtcttc accgtctaca acgagctcac gaaggtgaag tacgtcaccg 7020 agggcatgcg caagccagcg ttcctgtccg gggagcagaa gaaggctatc gtggacctcc 7080 tgttcagac caacggaag gtcacggtta agcactca ggaggactac ttcagaga 7140 tcgagtgctt cgattcggtc gagatcagcg gcgttgagga ccgcttcaac gccagcctcg 7200 ggacctacca cgatctcctg aagatcatca aggatagga cttcctggac aacgaggaga 7260 acgaggatat cctggaggac atcgtgctga cctcacgct gttcgaggac agggagatga 7320 tcgaggagcg cctgaagacg taccccatc tcttcgatga caggtcatg aagcaactca 7380 agccgccggag atacaccggc tgggggaggc tgtcccgcaa gctcatcac ggcatccggg 7440 acagcagtc cgggaagacc atcctcgact tcctcaagg cgatggctc gccaacagga 7500 acttcatgca actgatccac gatgacagcc tcaccttca ggaggatatc caaaaggctc 7560 aagtgagcgg ccagggggac tcgctgcacg agcatatcgc gaacctcgct ggctcccccg 7620 cgatcaagaa gggcatccctc cagaccgtga aggttgtgga cgagctcgtg aaggtcatgg 7680 gccggcacaa gcctgagaac atcgtcatcg agatggccag aggaaccaa accacgcaga 7740 aggggcaaaa gaactctagg gagcgcatga agcgcatcga ggagggcatc aaggagctgg 7800 ggtcccaaat cctcaaggag cacccagtgg agaacaccca actgcagaac gagaagctct 7860 acctgtacta cctccagaac ggcagggata tgtacgtgga ccaagagctg gatatcaacc 7920 gcctcagcga ttacgacgtc gatcatatcg ttccccagtc tttcctgaag gatgactcca 7980 tcgaacaaa ggtcctcacc aggtcggaca agaaccgcgg caagtcagat aacgttccat 8040 ctgaggaggt cgttaagaag atgaagaact actggaggca gctcctgaac gccaagctga 8100 tcacgcaaag gaagttcgac aacctcacca aggctgagag aggcgggctc tcagagctgg 8160 aaaggccgg cttcatcaag cggcagctgg tcgagaccag acaaatcacg aagcacgttg 8220 cgcaaatcct cgactctcgg atgaacacga agtacgatga gaacgacaag ctgatcaggg 8280 aggttaaggt gatcaccctg aagtctaagc tcgtctccga cttcaggaag gatttccagt 8340 tctacaaggt tcgcgagatc aacaactacc accatgccca tgacgcttac ctcaacgctg 8400 tggtcggcac cgctctgatc aagaagtacc caaagctgga gtccgagttc gtgtacgggg 8460 actacaaggt ttaccgatgtg cgcaagatga tcgccaagtc ggagcaagag atcggcaagg 8520 ctaccgccaa gtacttcttc tactcaaaca tcatgaactt cttcaagacc gagatcacgc 8580 tggccaacgg cgagatccgg aagagaccgc tcatcgagac caacggcgag acgggggaga 8640 tcgtgtggga caagggcagg gatttcgcaga ccgtccgcaa ggttctctcc atgccccagg 8700 tgaacatcgt caagaagacc gaggtccaaa cgggcgggtt ctcaaaggag tctatcctgc 8760 ctaagcggaa cagcgacaag ctcatcgcca gaagaagga ctgggaccca aagaagtacg 8820 gcgggtttcga cagccctacc gtggcctact cggtcctggt tgtggcgaag gttgagaagg 8880 gcaagtccaa gaagctcaag agcgtgaagg agctcctggg gatcaccatc atggagaggt 8940 ccagcttcga gaagaaccca atcgacttcc tggaggccaa gggctacaag gaggtgaaga 9000 aggacctgat catcaagctc ccgaagtact ctctcttga gctggagaac ggcaggaaga 9060 gaatgctggc ttccgctggc gagctccaga agggaacga gctcgcgctg ccaagcaagt 9120 9180 acgagcaaaa gcagctgttc gtcgagcagc acaagcatta cctcgacgag atcatcgagc 9240 aaatctccga gttcagcaag cgcgtgatcc tcgccgacgc gaacctggat aaggtcctct 9300 ccgcctacaa caagcaccgg gacaagccca tcagagagca agcggagaac atcatccatc 9360 tcttcaccct gacgaacctc ggcgctcctg ctgctttcaa gtacttcgac accacgatcg 9420 atcggaagag atacacctcc acgaaggagg tcctggacgc gaccctcatc caccagtcga 9480 tcaccggcct gtacgagacg agatcgacc tctcacaact cggcggggat aagagacccg 9540 cagcaaccaa gaaggcaggg caagcaaaga agaagaagac gcgtgactcc ggcggcagc 9599 <210> 7 <211> 1077 <212> DNA <213> Artificial Sequence <400> 7 atgtggtgca tgcggagact gcctacaaac agtgtgatga cagtggccag aaagcggaag 60 cagaccacca tcgaggactt ctttggcaca aagaagtcta ccaatgaggc ccctaacaag 120 aagggcaaga gcggcgctac attcatgacc atcacaaacg gcgccgccat taagaccgag 180 accaaggccg tggccaagga agccaacacc gacaagtatc ctgctaacag caacgccaag 240 gacgtgtaca gcaaaaacct gagcagcaat ctgagaaccc tgctttctct ggaactggaa 300 accatcgacg acagctggtt cccccacctg atggacgagt tcaagaaacc ctacttcgtg 360 aagctgaagc aatttgtgac aaaggagcag gccgaccaca ccgtgttccc tccagctaaa 420 gatatctaca gctggaccag actcacccct ttcaacaagg tgaaagtggt gatcatcggc 480 caggaccctt accacaactt caaccaggcc cacggcctg ccttcagcgt gaagcctcct 540 acccctgcc ctcctagcct gaagaacatc tacaaggaac tgaaacagga gtaccccgat 600 ttcgttgaag ataacaaggt gggcgatctg acccactggg ccagccaggg cgtgctgctc 660 ctgaacacct ccctgacagt gcgggcccat aatgccaatt cccacagcaa gcacggatgg 720 gagaccttca caaaaagagt ggtgcagctg ctgatccagg atagagaggc tgatggcaag 780 agcctggtgt tcctgctgtg gggcaacaac gccatcaagc tggtcgagag cctgctgggc 840 agcaccagcg ttggctctgg aagcaagtac cccaacatca tggtgatgaa aagcgtgcac 900 ccttctccac tgtccgcctc tagggattt tttggtacaa atcacttcaa gatgatcaac gactggctgt acaacacaag aggcgagaag atgatcgact ggtccgtggt ccccggcacc agcctgcggg aagtgcaaga ggccaacgct agctggaat ccgagtctaa agacccc 1077 <210> 8 <211> 4950 <212> DNA <213> Artificial Sequence <400> 8 ggtaccgatt agtgaacgga tctcgacggt atcgatcacg agactagcct cgagcggccg 120. cccccttcac cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga attack ctgtaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtattcga tttcttggct ttatatatct 360. tgtggaaagg acgaaacacc ggtcatctta gtcattacct ggttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttaaagaa ttctcgacct cgagacaaat ggcagtattc atccacaatt ttaaaagaaa 480 agggggggatt ggggggtaca gtgcaggga aagaatagta gacataatag caacagacat 540 acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag 600 ggacagcaga gatccacttt ggccgcggct cgaggggggtt ggggttgcgc cttttccaag 660 gcagccctgg gtttgcgcag ggacgcggct gctctgggcg tggttccggg aaacgcagcg 720 gcgccgaccc tgggactcgc acattcttca cgtccgttcg cagcgtcacc cggatcttcg 780 ccgctaccct tgtgggcccc ccggcgacgc ttcctgctcc gcccctaagt cgggaaggtt 840 ccttgcggtt cgcggcgtgc cggacgtgac aaacggaagc cgcacgtctc actagtaccc 900 tcgcagacgg acagcgccag ggagcaatgg cagcgcgccg accgcgatgg gctgtggcca 960 atagcggctg ctcagcaggg cgcgccgaga gcagcggccg ggaaggggcg gtgcgggagg 1020 cggggtgtgg ggcggtagtg tgggccctgt tcctgcccgc gcggtgttcc gcattctgca 1080 agcctccgga gcgcacgtcg gcagtcggct ccctcgttga ccgaatcacc gacctctctc 1140 cccaggggga tccaccggag cttaccatga ccgagtacaa gcccacggtg cgcctcgcca 1200 cccgcgacga cgtcccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactacccg 1260 ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac 1320 tcttctccac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg 1380 cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg 1440 gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc 1500 tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc 1560 ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg 1620 agcgcgccgg ggtgcccgcc ttcctggaaa cctccgcgcc ccgcaacctc cccttctacg 1680 agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt 1740 gcatgacccg caagcccggt gcctgacgcc cgccccacga cccgcagcgc ccgaccgaaa 1800 ggagcgcacg accccatgca tcggtacctt taagaccaat gacttacaag gcagctgtag 1860 atcttagcca ctttctagag tcggggcggc cggccgcttc gagcagacat gataagatac 1920 attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa 1980 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac 2040 aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt ttttaaagc 2100 aagtaaaacc tctacaaatg tggtaaaatc gataaggatc cgtcgaccga tgcccttgag 2160 agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact 2220 tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc tcttccgctt 2280 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 2340 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 2400 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg ttttccata 2460 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 2520 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 2580 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 2640 tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 2700 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 2760 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 2820 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 2880 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 2940 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 3000 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 3060 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 3120 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct 3180 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta 3240 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa 3300 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgggacccac 3360 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa 3420 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag 3480 tagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg 3540 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag 3600 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg 3660 tcaagagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc 3720 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat 3780 tctgagata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata 3840 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa 3900 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca 3960 actgatcttc agcatcttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc 4020 aaaatgccgc aaaaaaggga ataagggcga cacggaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caatagggg ttccgcgcac atttccccga aaagtgccac ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 4260 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg 4320 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat 4380 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagcg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata 4560. gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt father tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttacgcga ttttacaaa atattacgt ttacaatttc ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag cccaagctac 4740 catgataagt aagtaatatt aaggtacggg aggtacttgg agcggccgca ataaaatatc 4800 tttattttca ttacatctgt gtgttggttt tttgtgtgaa tcgatagtac taacatacgc 4860 tctccatcaa aacaaaacga aacaaaacaa actagcaaaa taggctgtcc ccagtgcaag 4920 tgcaggtgcc agaacatttc tctatcgata 4950 <210> 9 <211> 10526 <212> DNA <213> Artificial Sequence <400> 9 atgtggtgca tgcggagact gcctacaaac agtgtgatga cagtggccag aaagcggaag 60 cagaccacca tcgaggactt ctttggcaca aagaagtcta ccaatgaggc ccctaacaag 120 aagggcaaga gcggcgctac attcatgacc atcacaaacg gcgccgccat taagaccgag 180 accaaggccg tggccaagga agccaacacc gacaagtatc ctgctaacag caacgccaag 240 gacgtgtaca gcaaaaacct gagcagcaat ctgagaaccc tgctttctct ggaactggaa 300 accatcgacg acagctggtt cccccacctg atggacgagt tcaagaaacc ctacttcgtg 360 aagctgaagc aatttgtgac aaaggagcag gccgaccaca ccgtgttccc tccagctaaa 420 gatatctaca gctggaccag actcacccct ttcaacaagg tgaaagtggt gatcatcggc 480 caggaccctt accacaactt caaccaggcc cacggcctg ccttcagcgt gaagcctcct 540 acccctgcc ctcctagcct gaagaacatc tacaaggaac tgaaacagga gtaccccgat 600 ttcgttgaag ataacaaggt gggcgatctg acccactggg ccagccaggg cgtgctgctc 660 ctgaacacct ccctgacagt gcgggcccat aatgccaatt cccacagcaa gcacggatgg 720 gagaccttca caaaaagagt ggtgcagctg ctgatccagg atagagaggc tgatggcaag 780 agcctggtgt tcctgctgtg gggcaacaac gccatcaagc tggtcgagag cctgctgggc 840 agcaccagcg ttggctctgg aagcaagtac cccaacatca tggtgatgaa aagcgtgcac 900 ccttctccac tgtccgcctc taggggattt tttggtacaa atcacttcaa gatgatcaac 960 gactggctgt acaacacaag aggcgagaag atgatcgact ggtccgtggt ccccggcacc 1020 agcctgcggg aagtgcaaga ggccaacgct agactggaat ccgagtctaa agacccctcg 1080 ggggggagcc caaagaagaa gcggaaggtg tagtgtacaa gtaacccggg aattcggtac 1140 gctgaaatca ccagtctctc tctacaaatc tatctctctc tatttctcc ataaataatg 1200 tgtgagtagt ttcccgataa gggaaattag ggttcttata gggttcgct catgtgttga 1260 gcatataaga aacccttagt atgtatttgt atttgtaaaa tacttctatc aataaaattt 1320 ctaattccta aaaccaaaat ccagtactaa aatccagatc tcctaaagtc cctatagatc 1380 tttgtcgtga atataaacca gacacgagac gactaaacct ggagcccaga cgccgttcga 1440 agctagaagt accgcttagg caggaggccg ttagggaaaa gatgctaagg cagggttggt 1500 tacgttgact cccccgtagg tttggtttaa atatgatgaa gtggacggaa ggaaggagga 1560 agacaaggaa ggataaggtt gcaggccctg tgcaaggtaa gaagatggaa atttgataga 1620 ggtacgctac tatacttata ctatacgcta agggaatgct tgtatttata ccctataccc 1680 cctaataacc ccttatcaat ttaagaaata atccgcataa gccccgctt aaaaattggt 1740 atcagagcca tgaataggtc tatgaccaaa actcaagagg ataaaacctc accaaaatac 1800 gaaagagttc ttaactctaa agataaaaga tctttcaaga tcaaaactag ttccctcaca 1860 ccggtgacgg ggatcgcatg cgatatctcg agatctagct tggcgtaatc atggtcatag 1920 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc 1980 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc 2040 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa 2100 cgcgcgggga gaggcggttt gcgtattggg cggccttccg cttcctcgct cactgactcg 2160 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 2220 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag 2280 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 2340 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 2400 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 2460 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc 2520 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 2580 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 2640 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 2700 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctcac tagagaaca 2760 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 2820 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 2880 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 2940 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc 3000 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa 3060 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta 3120 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc 3180 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat 3240 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta 3300 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt 3360 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt 3420 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg 3480 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc 3540 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc 3600 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg 3660 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga 3720 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta 3780 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct 3840 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag 3900 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga 3960 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat 4020 aaaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgccag tgccaagcta 4080 attcgagctc ggtacctgac ccggtcgtgc ccctctctag agataatgag cattgcatgt 4140 ctaagttata aaaattacc acatattttt tttgtcacac ttgtttgaag tgcagtttat 4200 ctatctttat acatatattt aaactttact ctacgaataa tataatctat agtactacaa 4260 tatatcagt gttttagaga atcatataaa tgaacagtta gacatggtct aaaggacaat 4320 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga 4380 tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta 4440 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc 4500 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg 4560 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg 4620 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa 4680 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac 4740 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg 4800 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg 4860 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca 4920 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg 4980 ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag 5040 acccaagctg gctagcgttt aaacttaagc ttgtcgacgg atccatgcca aagaagaaga 5100 ggaaggtttc atcggagacc ggccctgttg ctgttgaccc caccctgcgg cggagaatcg 5160 agccacacga gttcgaggtg ttcttcgacc caagggagct cgccaaggag acgtgcctcc 5220 tgtacgagat caactggggc ggcaggcact ccatctggag gcacaccagc caaaacacca 5280 acaagcacgt ggaggtcaac ttcatcgaga agttcaccac cgagaggtac ttctgcccaa 5340 acacccgctg ctccatcacc tggttcctgt cctggagccc atgcggcgag tgctccaggg 5400 ccatcaccga gttcctcagc cgctacccac acgtcaccct gttcatctac atcgccaggc 5460 tctaccacca cgccgaccca aggaacaggc agggcctccg cgacctgatc tccagcggcg 5520 tgaccatcca aatcatgacc gagcaggagt ccggctactg ctggaggaac ttcgtcaact 5580 actccccaag caacgaggcc cactggccaa ggtacccaca cctctgggtg cgcctctacg 5640 tgctcgagct gtactgcatc atcctcggcc tgccaccatg cctcaacatc ctgaggcgca 5700 agcaaccaca gctgaccttc ttcaccatcg ccctccaaag ctgccactac cagaggctcc 5760 caccacacat cctgtgggct accggcctca agtccggcag cgagacgcca ggcacctccg 5820 agagcgctac gcctgaactt aaggacaaga agtactcgat cggcctcgcc atcgggacga 5880 actcagttgg ctgggccgtg atcaccgacg agtacaaggt gccctctaag aagttcaagg 5940 tcctggggaa caccgaccgc cattccatca agaagaacct catcggcgct ctcctgttcg 6000 acagcgggga gaccgctgag gctacgaggc tcaagagaac cgctaggcgc cggtacacga 6060 gaaggaagaa caggatctgc tacctccaag agattttctc caacgagatg gccaaggttg 6120 acgattcatt cttccaccgc ctggaggagt cttcctcgt ggaggaggat aagaagcacg 6180 agcggcatcc catcttcggc aacatcgtgg acgaggttgc ctaccacgag aagtacccta 6240 cgatctacca tctgcggaag aagctcgtgg actccaccga taaggcggac ctcagactga 6300 tctacctcgc tctggcccac atgatcaagt tccgcggcca tttcctgatc gagggggatc 6360 tcaacccaga caacagcgat gttgacaagc tgttcatcca actcgtgcag acctacaacc 6420 aactcttgga ggagaacccg atcaacgcct ctggcgtgga cgcgaaggct atcctgtccg 6480 cgaggctctc gaagtccagg aggctggaga acctgatcgc tcagctccca ggcgaagaga 6540 agaacggcct gttcgggaac ctcatcgctc tcagcctggg gctcaccccg aacttcaagt 6600 cgaacttcga tctcgctgag gacgccaagc tgcaactctc caaggacacc tacgacgatg 6660 acctcgataa cctcctggcc cagatcggcg atcaatacgc ggacctgttc ctcgctgcca 6720 agaacctgtc ggacgccatc ctcctgtcag atatcctccg cgtgaacacc gagatcacga 6780 aggctccact ctctgcctcc atgatcaagc gctacgacga gcaccatcag gatctgaccc 6840 tcctgaaggc gctggtccgc caacagctcc cggagaagta caaggagatt ttcttcgatc 6900 agtcgaagaa cggctacgct gggtacatcg acggcggggc ctcacaagag gagttctaca agttcatcaa gccaatcctg gagaagatgg acggcacgga ggagctcctg gtgaagctca acagggagga cctcctgcgg aagcagagga ccttcgataa cggcagcatc ccccaccaaa tccatctcgg ggagctgcac gccatcctga gaaggcaaga ggacttctac cctttcctca aggataaccg ggagaagatc gagaagatcc tgaccttcag aatcccatac tacgtcggcc ctctcgcgcg ggggaactca agattcgctt ggatgacccg caagtctgag gagaccatca cgccgtggaa cttcgaggag gtggtggaca agggcgctag cgctcagtcg ttcatcgaga 7320 ggatgaccaa cttcgacaag aacctgccca acgagaaggt gctccctaag cactcgctcc tgtacgagta cttcaccgtc tacaacgagc tcacgaaggt gaagtacgtc accgagggca tgcgcaagcc agcgttcctg tccggggagc agaagaaggc tatcgtggac ctcctgttca 7500. agccaaccg gaaggtcacg gttaagcaac tcaaggagga ctacttcaag aagatcgagt gcttcgattc ggtcgagatc agcggcgttg aggaccgctt caacgccagc ctcgggacct 7620 accacgatct cctgaagatc atcaaggata aggacttcct ggacaacgag gagaacgagg 7680 atatcctgga ggacatcgtg ctgaccctca cgctgttcga ggacagggag atgatcgagg 7740 agcgcctgaa gacgtacgcc catctcttcg atgacaaggt catgaagcaa ctcaagcgcc 7800 ggagatacac cggctggggg aggctgtccc gcaagctcat caacggcatc cgggacaagc 7860 agtccgggaa gaccatcctc gacttcctca agagcgatgg cttcgccaac aggaacttca 7920 tgcaactgat ccacgatgac agcctcacct tcaaggagga tatccaaaag gctcaagtga 7980 gcggccaggg ggactcgctg cacgagcata tcgcgaacct cgctggctcc cccgcgatca 8040 agaagggcat cctccagacc gtgaaggttg tggacgagct cgtgaaggtc atgggccggc 8100 acaagcctga gaacatcgtc atcgagatgg ccagagagaa ccaaaccacg cagaaggggc 8160 aaaagaactc tagggagcgc atgaagcgca tcgaggaggg catcaaggag ctggggtccc 8220 aaatcctcaa ggagcaccca gtggagaaca cccaactgca gaacgagaag ctctacctgt 8280 actacctcca gaacggcagg gatatgtacg tggaccaaga gctggatatc aaccgcctca 8340 gcgattacga cgtcgatcat atcgttcccc agtctttcct gaaggatgac tccatcgaca 8400 acaaggtcct caccaggtcg gacaagaacc gcggcaagtc agataacgtt ccatctgagg 8460 aggtcgttaa gaagatgaag aactactgga ggcagctcct gaacgccaag ctgatcacgc 8520 aaaggaagtt cgacaacctc accaaggctg agagaggcgg gctctcagag ctggacaagg 8580 ccggcttcat caagcggcag ctggtcgaga ccagacaaat cacgaagcac gttgcgcaaa 8640 tcctcgactc tcggatgaac acgaagtacg atgagaacga caagctgatc agggaggtta 8700 aggtgatcac cctgaagtct aagctcgtct ccgacttcag gaaggatttc cagttctaca 8760 aggttcgcga gatcaacaac taccaccatg cccatgacgc ttacctcaac gctgtggtcg 8820 gcaccgctct gatcaagaag tacccaaagc tggagtccga gttcgtgtac ggggactaca 8880 aggtttacga tgtgcgcaag atgatcgcca agtcggagca agagatcggc aaggctaccg 8940 ccaagtactt cttctactca aacatcatga acttcttcaa gaccgagatc acgctggcca 9000 acggcgagat ccggaagaga ccgctcatcg agaccaacgg cgagacgggg gagatcgtgt 9060 gggacaaggg cagggatttc gcgaccgtcc gcaaggttct ctccatgccc caggtgaaca 9120 tcgtcaagaa gaccgaggtc caaacgggcg ggttctcaaa ggagtctatc ctgcctaagc 9180 ggaacagcga caagctcatc gccagaaaga agactggga cccaaagaag tacggcgggt 9240 tcgacagccc taccgtggcc tactcggtcc tggttgtggc gaaggttgag aagggcaagt 9300 ccaagaagct caagagcgtg aaggagctcc tggggatcac catcatggag aggtccagct 9360 tcgagaagaa cccaatcgac ttcctggagg ccaagggcta caaggaggtg aagaaggacc 9420 tgatcatcaa gctcccgaag tactctctct tcgagctgga gaacggcagg aagagaatgc 9480 tggcttcgc tggcgagctc cagaagggga acgagctcgc gctgccaagc aagtacgtga 9540 acttcctcta cctggcttcc cactacgaga agctcaaggg cagcccggag gacaacgagc 9600 aaaagcagct gttcgtcgag cagcacaagc attacctcga cgagatcatc gagcaaatct 9660 ccgagttcag caagcgcgtg atcctcgccg acgcgaacct ggataaggtc ctctccgcct 9720 acaacaagca ccgggacaag cccatcagag agcaagcgga gaacatcatc catctcttca 9780 ccctgacgaa cctcggcgct cctgctgctt tcaagtactt cgacaccacg atcgatcgga 9840 agatacac ctccacgaag gaggtcctgg acgcgaccct catccaccag tcgatcaccg 9900 gcctgtacga gacgaggatc gaccctcac aactcggcgg ggataagaga cccgcagcaa 9960 ccaagaaggc agggcaagca aagagaga agagcggagg atctagcgga ggatcaagcg 10020 gaagcgagac tcctggaacc agcgaagcg haaccccaga aagcagcgga ggagtagcg 10080 gaggaagcat ggcaatgcag atgcagcttg aagcaatgc agatacttca gtggaagaag 10140 aaagctttgg cccacaaccc atttcacggt gccacacggt gccacataat gccacggtg 10200 tgaagaattt ggaagaagct ggattccata ctgtggaggc tgttgcctat gcgccaaaga 10260 aggagctaat aaatattaag ggaattagtg agccaagc tgataaatt ctggctgagg 10320 cagctaaatt agttccaatg gtttcacca ctgcaactga attccacca aggcggtcag 10380 agatcataca gattactact gggctccaag agcttgacaa actactca tctggcggat 10440 ctagcggtgg atctagcggc tctgagaccc ctggaacatc cgaatccgcc actcagaga 10500 gcagcggagg ctcttctgga ggatca <210> 10 <211> 750 <212> DNA <213> Artificial Sequence <400> 10 atgaccaaca ccctgtggaa cagcgtggac gagctgccca tccacgacag ctggaagccc gtgctgaagc ccgtggagga cgccatcaga aagctgggcg tgttcctggc cgaggaggag 120 ttcctgcccc ccgtggacga cgtgttcaga gccttcagct accccttcga cgccgtgaag gtactgatca tgggccaaga cccctacccc acccccggcc atgccatggg cctgagcttc 240 agcacacagc ccgacgtcag acccctgcct accacctga acaacatctt caaagaactg gtgagcgacg tcggaagcct gggcgatagc gcgtccgagc aaggcgccct ggacctggga 360 atcaacgcgc cgggctccgt ggctgggacc caagttgccc tgcccgctga tggcgacctg 420 agagcctgga gcaaccaagg cgtggccctg ttcaacagag tactgaccgt gcatcccggc 480 caagccggca gccataaagg caagggctgg gaggccgtga ccgagcaagc catcaaggcc ctggccgaga gagatcagcc cctggtggcc atcctgtggg gcaagcaagc ccaagaggtg cagaagttcc tgggcgacac cccctgcatc tgcagcgtgc accctagccc cctgagcgct 660 agcagaggct tcttcggcag caagcccttc agcagagcca acgagatcct gtcaagcctg 720 ggcgccaccg agatcgactg gagcctgtga 750

Claims

1. Base editing system, which is the base editing system APOBEC-nCas9-Rad51-Ung1; The base editing system APOBEC-nCas9-Rad51-Ung1 includes cytosine deaminase APOBEC or biological materials related thereto, nuclease nCas9 or biological materials related thereto, single-stranded DNA binding protein Rad51 or biological materials related thereto, and uracil DNA glycosylase Ung1 or biological materials related thereto; The nuclease nCas9 is C1) or C2): C1) The amino acid sequence is the protein shown in Sequence 2; C2) A fusion protein obtained by connecting a tag to the N-terminus or / and the C-terminus of C1); The biological material associated with the nuclease nCas9 is any one of D1) to D4): D1) a nucleic acid molecule encoding the nCas9 nuclease; D2) an expression cassette containing the nucleic acid molecule described in D1); D3) a recombinant vector containing the nucleic acid molecule described in D1); D4) a transgenic cell line containing the nucleic acid molecule described in D1); The uracil DNA glycosylase Ung1 is E1) or E2): E1) The amino acid sequence is the protein shown in SEQ ID NO:3; E2) A fusion protein obtained by connecting a tag to the N-terminus or / and C-terminus of E1); The biological material related to the uracil DNA glycosylase Ung1 is any one of F1) to F4): F1) a nucleic acid molecule encoding the uracil DNA glycosylase Ung1; F2) an expression cassette containing the nucleic acid molecule described in F1); F3) a recombinant vector containing the nucleic acid molecule described in F1); F4) a transgenic cell line containing the nucleic acid molecule described in F1); The cytosine deaminase APOBEC is G1) or G2): G1) The amino acid sequence is the protein shown in SEQ ID NO:4; G2) A fusion protein obtained by connecting a tag to the N-terminus or / and C-terminus of G1); The biological material associated with the cytosine deaminase APOBEC is any one of H1) to H4): H1) a nucleic acid molecule encoding the cytosine deaminase APOBEC; H2) an expression cassette containing the nucleic acid molecule described in H1); H3) a recombinant vector containing the nucleic acid molecule described in H1); H4) a transgenic cell line containing the nucleic acid molecule described in H1); The single-stranded DNA binding protein Rad51 is I1) or I2): I1) The amino acid sequence is the protein shown in SEQ ID NO:5; I2) A fusion protein obtained by connecting a tag to the N-terminus or / and C-terminus of I1); The biological material related to the single-stranded DNA binding protein Rad51 is any one of J1) to J4): J1) a nucleic acid molecule encoding the single-stranded DNA binding protein Rad51; J2) an expression cassette containing the nucleic acid molecule described in J1); J3) a recombinant vector containing the nucleic acid molecule described in J1); J4) A transgenic cell line containing the nucleic acid molecule described in J1).

2. The base editing system according to claim 1, characterized in that: The base editing system also includes gRNA or biological materials related thereto; The biological material associated with the gRNA is any one of K1) to K4): K1) a nucleic acid molecule for transcribing the gRNA; K2) an expression cassette containing the nucleic acid molecule described in K1); K3) a recombinant vector containing the nucleic acid molecule described in K1); K4) A transgenic cell line containing the nucleic acid molecule described in K1).

3. Use of the base editing system of claim 1 or 2 for non-disease diagnosis and treatment purposes in any one of X1) to X6): X1) Base editing of the genome sequence of an organism or biological cell; X2) Preparation of base editing products of the genome sequence of organisms or biological cells; X3) Improving the efficiency of base editing of genome sequences of organisms or biological cells; X4) Preparation of products that improve the efficiency of base editing of genomic sequences of organisms or biological cells; X5) Improving the purity of base editing products of the genome sequence of organisms or biological cells; X6) Preparation of products that improve the purity of base editing products of the genome sequence of organisms or biological cells; The organism is a mammal; the biological cell is a mammalian cell; The base editing is from base C to base G.

4. A method for non-disease diagnosis and treatment purposes as described in any one of the following Y1) to Y3): Y1) A method for base editing of a genomic sequence, comprising the following steps: introducing the cytosine deaminase APOBEC, the nuclease nCas9, the single-stranded DNA binding protein Rad51, the uracil DNA glycosylase Ung1 and the gRNA described in claim 2 into an organism or a biological cell to achieve base editing; Y2) A method for improving the base editing efficiency of a genome sequence of an organism or a biological cell, comprising the following steps: introducing the cytosine deaminase APOBEC, the nuclease nCas9, the single-stranded DNA binding protein Rad51, the uracil DNA glycosylase Ung1 and the gRNA described in claim 2 into the organism or the biological cell to improve the base editing efficiency; Y3) A method for improving the purity of a base editing product of a genome sequence of an organism or a biological cell, comprising the following steps: introducing the cytosine deaminase APOBEC, the nuclease nCas9, the single-stranded DNA binding protein Rad51, the uracil DNA glycosylase Ung1 and the gRNA described in claim 2 into an organism or a biological cell to improve the purity of the base editing product; The organism is a mammal; the biological cell is a mammalian cell; The base editing is from base C to base G.

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