CN112538471B

CN112538471B - CRISPR SpCas9 (K510A) mutant and application thereof

Info

Publication number: CN112538471B
Application number: CN202011587591.9A
Authority: CN
Inventors: 李娟�; 王国华; 王灿茂
Original assignee: Southern Medical University
Current assignee: Southern Medical University
Priority date: 2020-12-28
Filing date: 2020-12-28
Publication date: 2023-12-12
Anticipated expiration: 2040-12-28
Also published as: CN112538471A

Abstract

The invention discloses a CRISPR SpCas9 (K510A) mutant and application thereof, wherein the SpCas9 mutant is designed by mutating a 510 th lysine residue of wild SpCas9 into an alanine residue, wherein the amino acid sequence of the wild SpCas9 is shown as SEQ ID NO.1, and the amino acid sequence of the SpCas9 mutant is shown as SEQ ID NO. 2. The SpCas9 (K510A) mutant can remarkably reduce the editing efficiency of off-target sites, namely remarkably reduce the off-target rate under the condition that the shearing efficiency of the off-target sites is basically the same as that of the wild SpCas9.

Description

CRISPR SpCas9 (K510A) mutant and application thereof

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a CRISPR SpCas9 (K510A) mutant and application thereof.

Background

SpCas9 (SpCas 9) is widely used in gene editing. The SpCas9 gene editing system includes SpCas9 protein and sgrnas. The SpCas9 protein and sgRNA bind to form a complex that specifically recognizes the target site through a guide sequence on SpCas9 with the PAM interaction domain and the sgRNA, and double-stranded blunt-ended cleavage of the target DNA using the HNH domain and RuvC domain. In living cells, the sheared genomic DNA may initiate repair of NHEJ, resulting in insertion/deletion mutations (indels).

However, in the related art, although the wild type SpCas9 can efficiently edit the gene, some non-target sites similar to the target sites are edited at the same time of editing the target sites, which seriously affects the clinical application of the SpCas9.

Therefore, there is a need to develop a new SpCas9.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, compared with the conventional wild-type SpCas9, the SpCas9 mutant or the active fragment thereof provided by the invention can remarkably reduce the editing efficiency of off-target sites, namely remarkably reduce the off-target rate under the condition that the shearing efficiency of the target sites is basically the same as that of the wild-type SpCas9.

The invention also provides a gene for encoding the SpCas9 mutant or the active fragment thereof.

The invention also provides a composition.

The invention also provides a polynucleotide.

The invention also proposes a guide-polynucleotide/Cas complex.

The invention also provides a recombinant vector, recombinant bacteria or cells containing the genes.

The invention also provides a method for modifying a target site in a genome of a cell.

The invention also provides application of the SpCas9 mutant or the active fragment thereof or the gene in gene editing.

According to a first aspect of the present invention there is provided a SpCas9 mutant or an active fragment thereof, the SpCas9 mutant or active fragment thereof comprising: has at least 90% amino acid identity with the wild-type SpCas9 polypeptide shown in SEQ ID No.1, and the amino acid residue at position 510 of the REC3 domain of the wild-type SpCas9 polypeptide is mutated to an alanine residue, wherein the SpCas9 mutant has endonuclease activity.

According to a preferred embodiment of the invention, there is at least the following advantageous effect: the SpCas9 (K510A) mutant is designed by mutating the 510 th lysine residue of the wild SpCas9 (SEQ ID NO. 1) into an alanine residue. The inventor analyzes the interaction between the amino acid residue of the wild SpCas9 REC3 structural domain and the sgRNA/target DNA heterozygous double chain, discovers that a part of amino acid residues in the REC3 structure and the sgRNA/target DNA double chain form nonspecific hydrogen bond interaction, and obtains the hydrophilic amino acid residue Lysine residue (Lysine residue) at the 510 th position in the REC3 structure as an optimal mutation point after further screening the interaction force type, size and structure. By mutating the lysine residue at position 510 of the wild-type SpCas9 REC3 domain to an alanine residue, repeated verification demonstrates that the SpCas9 (K510A) mutant is able to significantly reduce the editing efficiency of off-target sites, i.e. significantly reduce the off-target rate, while maintaining substantially the same cleavage efficiency of the target sites as the wild-type SpCas9.

Wherein, the amino acid sequence of the wild SpCas9 shown in the SEQ ID NO.1 is as follows:

MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGSPKKKRKVSSDYKDHDGDYKDHDIDYKDDDDKAAG(SEQ ID NO.1)。

the bolded and underlined portion is the lysine residue before mutation;

only the underlined parts are modified sequences.

In some embodiments of the invention, the SpCas9 mutant or active fragment thereof described above further includes: has at least 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75% amino acid identity with the wild-type SpCas9 polypeptide shown in SEQ ID No.1, and the amino acid residue at position 510 of the REC3 domain of the wild-type SpCas9 polypeptide is mutated to an alanine residue, wherein the SpCas9 mutant has endonuclease activity.

In some embodiments of the invention, the amino acid sequence of the SpCas9 mutant or active fragment thereof described above includes:

(1) An amino acid sequence shown in SEQ ID NO. 2; or (b)

(2) The amino acid sequence of the SpCas9 (K510A) mutant shown in SEQ ID NO.2 is a sequence with endonuclease activity after being substituted, deleted and/or added with one or more amino acids and/or terminal modification.

Wherein, the amino acid sequence of the SpCas9 (K510A) mutant shown in the SEQ ID NO.2 is as follows:

MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPAHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGSPKKKRKVSSDYKDHDGDYKDHDIDYKDDDDKAAG(SEQ ID NO.2)。

the part which is thickened and marked with a lower slide wire is the alanine residue after mutation;

only the underlined parts are modified sequences.

In some preferred embodiments of the invention, the amino acid sequence of the SpCas9 mutant or active fragment thereof described above further comprises: and adding a nuclear signal peptide, a 3×flag or his6 tag or other modified sequences conventional in the art on the basis of the amino acid sequence shown in SEQ ID NO. 2.

According to a second aspect of the invention there is provided a gene encoding a SpCas9 mutant or an active fragment thereof according to the first aspect of the invention.

According to a preferred embodiment of the invention, there is at least the following advantageous effect: the above-mentioned gene encoding the SpCas9 mutant or the active fragment thereof according to the first aspect of the present invention may be used to express the SpCas9 mutant or the active fragment thereof according to the first aspect of the present invention, and the SpCas9 (K510A) mutant obtained by expression may significantly reduce the editing efficiency of off-target sites, that is, significantly reduce the off-target rate, while maintaining substantially the same cleavage efficiency of the target sites as that of the wild-type SpCas9.

According to a third aspect of the present invention there is provided a composition comprising a SpCas9 mutant or an active fragment thereof according to the first aspect of the present invention.

According to a preferred embodiment of the invention, there is at least the following advantageous effect: the composition contains the SpCas9 mutant or the active fragment thereof according to the first aspect of the invention, and the off-target rate in the process of gene editing can be remarkably reduced by using the composition.

According to a fourth aspect of the present invention there is provided a polynucleotide comprising a gene according to the second aspect of the present invention.

In some preferred embodiments of the invention, the polynucleotide is a guide-polynucleotide.

In some more preferred embodiments of the invention, the guide-polynucleotide is sgRNA.

In some more preferred embodiments of the present invention, the guide sequence (guide sequence) of the sgrnas described above is:

5’-gagtccgagcagaagaagaa-3’(SEQ ID NO.3)。

the targeting specificity of Cas9 depends on the presence of 20nt of the sgRNA guide sequence and PAM adjacent to the target sequence in the genome, thus, a rational and efficient involvement of the sgRNA guide sequence can effectively improve the usability of Cas9.

The sgRNA plasmid vector constructed using the guide sequence of the above sgrnas may also be inserted with an antibiotic resistance gene and a reporter Group (GFP) sequence.

In some more preferred embodiments of the invention, the antibiotic resistance gene comprises a eukaryotic puromycin resistance gene or a hygromycin resistance gene.

In some more preferred embodiments of the invention, the antibiotic resistance gene is a eukaryotic puromycin resistance gene.

In some more preferred embodiments of the invention, the reporter group comprises an EGFP group.

Of course, the person skilled in the art can insert other functional genes, such as an inducible promoter of doxycycline (doxycycline), to enhance the transient expression of Cas9 according to the actual needs.

According to a fifth aspect of the present invention there is provided a guide-polynucleotide/Cas complex comprising at least one guide-polynucleotide and at least one SpCas9 mutant or active fragment thereof according to the first aspect of the present invention;

wherein the guide-polynucleotide is a chimeric non-naturally occurring guide-polynucleotide;

the guide-polynucleotide/Cas complex is capable of recognizing, in whole or in part, binding to a target sequence and nicking or unwinding the target sequence, cleaving the target sequence.

In some more preferred embodiments of the present invention, the guide sequence of the above sgrnas is shown in SEQ ID No. 3.

The introduction of the CRISPR/Cas9 component into a cell is typically accomplished using a DNA introduction system, such as transfection of plasmids encoding Cas9 and sgrnas into the cell. However, when plasmid introduction is problematic, gene editing can be achieved by direct introduction of Cas9/sgRNA Ribonucleoprotein (RNP) complex or using viral vectors (e.g., lentiviral vectors). In contrast to the method of introducing plasmids, editing by the method of introducing Cas9/sgRNA Ribonucleoprotein (RNP) complex can be started quickly after introducing cells. The Cas9/sgRNA Ribonucleoprotein (RNP) complex has less off-target and less immunogenicity, and is more efficient and long lasting when transfected with viral vectors.

According to a sixth aspect of the present invention there is provided a recombinant vector, recombinant bacterium or cell comprising a gene according to the second aspect of the present invention.

In some embodiments of the invention, the cells comprise prokaryotic cells or eukaryotic cells.

In some preferred embodiments of the invention, the cells comprise: cells of animal, bacterial, fungal, insect, yeast and plant origin.

In some more preferred embodiments of the invention, the above-described cells comprise: human cells, animal cells, plant cells and unicellular organisms in vivo, ex vivo or in vitro; wherein the human cells and animal cells are preferably human and animal blood cells.

According to a seventh aspect of the present invention there is provided a method of modifying a target site in a genome of a cell, comprising: introducing the guide-polynucleotide/Cas complex of the fifth aspect of the invention into a cell such that the target site in the cell has the following modifications:

substitution of at least one nucleotide; and/or

A deletion of at least one nucleotide; and/or

Insertion of at least one nucleotide.

According to an eighth aspect of the present invention there is provided the use of the SpCas9 mutant or the active fragment thereof according to the first aspect of the present invention or the gene according to the second aspect of the present invention in gene editing.

According to a preferred embodiment of the invention, there is at least the following advantageous effect: the SpCas9 (K510A) mutant is designed by mutating the 510 th lysine residue of the wild SpCas9 into alanine residue. The inventor analyzes the interaction between the amino acid residue of the wild SpCas9 REC3 structural domain and the sgRNA/target DNA heterozygous double chain, discovers that a part of amino acid residues in the REC3 structure and the sgRNA/target DNA double chain form nonspecific hydrogen bond interaction, and obtains the hydrophilic amino acid residue Lysine residue (Lysine residue) at the 510 th position in the REC3 structure as an optimal mutation point after further screening the interaction force type, size and structure. By mutating the lysine residue at position 510 of the wild-type SpCas9 REC3 domain to an alanine residue, repeated verification demonstrates that the SpCas9 mutant SpCas9 (K510A) is capable of significantly reducing the editing efficiency of off-target sites, i.e. significantly reducing the off-target rate, while maintaining substantially the same cleavage efficiency of the target sites as the wild-type SpCas9.

Drawings

The invention is further described with reference to the accompanying drawings and examples, in which:

FIG. 1 is a plasmid map of SpCas9 in an embodiment of the invention;

FIG. 2 is a map of an sgRNA plasmid in an example of the present invention;

FIG. 3 is a two-dimensional graph showing the editing efficiency results of wild SpCas9 at three sites of EMX1-1 (A), EMX1-1-OT1 (B) and EMX1-1-OT2 (C); wherein the blue point: single FAM positive, green dot: single VIC positive, grey dot: FAM/VIC double negative, brown dot: FAM/VIC double positive;

FIG. 4 is a two-dimensional graph showing the results of editing efficiency of mutant SpCas9 (K510A) at three sites of EMX1-1 (A), EMX1-1-OT1 (B) and EMX1-1-OT2 (C); wherein the blue point: single FAM positive, green dot: single VIC positive, grey dot: FAM/VIC double negative, brown dot: FAM/VIC double positive.

Detailed Description

The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.

Terms in the specification regarding the present invention: spCas9 refers to streptococcus pyogenes (Streptococcus pyogenes) Cas9, cas refers to clustered regularly interspaced palindromic sequences (CRISPR (clustered regularly interspaced short palindromic repeats) associded).

The term "Cas protein" or "Cas polypeptide" refers to a polypeptide encoded by a Cas (CRISPR-associated) gene. Cas proteins include Cas endonucleases.

The term "Cas endonuclease" refers to a Cas polypeptide (Cas protein) that is capable of recognizing, binding to, and optionally nicking or cleaving all or part of a specific DNA target sequence when complexed with a suitable polynucleotide component. Cas endonucleases are directed by the guide-polynucleotides to recognize, bind to, and optionally nick or cleave all or part of a particular target site in double-stranded DNA (e.g., at a target site in the genome of a cell). The Cas endonucleases described herein comprise one or more nuclease domains. Cas endonucleases employed in the donor DNA insertion methods described herein are endonucleases that introduce single-or double-strand breaks into DNA at a target site. Alternatively, cas endonucleases herein may lack DNA cleavage or nicking activity, but may still specifically bind to DNA target sequences when complexed with suitable RNA components.

Plasmid design and construction

In the following examples, the BPK4410 plasmid (Addgene plasmid # 101178) is taken as an example, but it should be noted that the present invention is not limited to BPK4410 plasmid, and alternative plasmids can be reasonably selected as experimental vectors within the knowledge of one of ordinary skill in the art.

(1) SpCas9 mutant plasmid vector construction:

the inventors hypothesize that compaction of the sgRNA/target DNA hybrid duplex may cause an increase in the hydrophobicity of the duplex, thereby causing an increase in the hydrophobic interaction between the sgRNA/target DNA hybrid duplex and the REC3 domain, and that the increased hydrophobic interaction results in movement of the REC3 domain towards the sgRNA/target DNA hybrid duplex, thereby initiating cleavage. It is further envisaged that the best case of the above findings should be that the forces between the REC3 domain and the sgRNA/target DNA hybrid duplex are only hydrophobic interactions without non-specific interactions such as hydrogen bonds, and that movement of the REC3 domain to the sgRNA/target DNA hybrid duplex is only initiated when the sgRNA is 100% matched to the target DNA strand. According to the idea, the inventor finds that part of amino acid residues form nonspecific hydrogen bond interactions with the sgRNA/target DNA double strand by analyzing interactions between the amino acid residues of the REC3 domain and the sgRNA/target DNA double strand, and further obtains the optimal mutant SpCas9 (K510A) by screening and obtaining the hydrophilic amino acid residue lysine residue at the position 510 through mutation into alanine residue by taking factors such as the type, the size and the structure of interaction force and the like into consideration.

The concrete construction steps are as follows:

taking plasmid BPK4410 (hypersas 9, edge plasmid # 101178) as a template, firstly mutating the alanine residue at 692 position of hypersas 9 into an asparagine residue, mutating the alanine residue at 694 into a methionine residue, mutating the alanine residue at 695 into a glutamine residue, mutating the alanine residue at 698 into a histidine residue, and obtaining wild-type SpCas9.

Mutation of lysine residue at position 510 of the SpCas9 amino acid sequence to alanine residue resulted in mutant SpCas9 (K510A).

The amino acid sequence of the wild SpCas9 is shown as SEQ ID NO.1, and the amino acid sequence of the mutant SpCas9 (K510A) after mutation is shown as SEQ ID NO. 2.

The nucleotide sequence of the plasmid expressing wild SpCas9 is shown as SEQ ID NO. 4.

The nucleotide sequence of the plasmid expressing SpCas9 (K510A) is shown in SEQ ID NO. 5:

the plasmid map of the constructed SpCas9 (K510A) mutant is shown in figure 1.

(2) Construction of sgRNA plasmid vector (sgRNA-EMX 1-1-puro):

the sgRNA is designed, and after screening, the sequence shown in SEQ ID NO.3 is selected as a guide sequence.

The sequence shown in SEQ ID NO.3 is:

5’-gagtccgagcagaagaagaa-3’(SEQ ID NO.3)。

the constructed sgRNA plasmid vector also comprises a promoter, a eukaryotic puromycin resistance gene sequence and an EGFP reporter gene sequence, and can express green fluorescent protein after transfection, and can use puromycin to carry out medicine screening to screen positive cells successfully transfected.

To verify the actual effect of the SpCas9 mutant, the EMX1-1 site of the EMX1 gene was used as a target site, and the sgRNA plasmid vector, designated as sgRNA-EMX1-1-puro, was targeted to the target site.

The nucleotide sequence of the constructed sgRNA plasmid vector (sgRNA-EMX 1-1-puro) is shown as SEQ ID NO.6, and the plasmid map is shown as FIG. 2.

The nucleotide sequence of the plasmid expressing sgRNA-EMX1-1 is shown as SEQ ID NO. 6).

Cell culture

HEK293T cells were used as transfection targets.

HEK293T cells were cultured in DMEM medium (10% FBS, 2mM glutamine and two antibiotics (penicillin and streptomycin)) at 37℃under 5% CO ₂ 。

Construction of transfected cells

HEK293T cells cultured in the above examples were seeded into 24-well plates (2X 10) ⁵ Individual cells/well), 0.5ml of complete medium was added to each well and incubated overnight. Transfection was performed until the next day at a cell fusion of about 70-80%. Transfection cells were transfected using a total of 1 μg of plasmid (750 ng SpCas9/SpCas9 (K510A) plasmid and 250ng sgRNA-EMX1-1-puro plasmid) using Lipofectamine 3000 (Invitrogen, 1.5 μl lipo3000,2 μ L p 3000) as transfection reagent, reference reagent instructions or routine procedures in the art.

The transfected cells were screened with 2. Mu.g/mL Puromycin on days 2-4 after transfection and genomic DNA was extracted the next day after screening was completed for use.

Editing efficiency detection at target site and off-target site

The sites EMX1-1-OT1 and EMX1-1-OT2 with higher off-target rate of the wild SpCas9 when the EMX1-1 site is sheared are selected, which are confirmed by the report, and are used for detecting off-target conditions of SpCas9 (K510A) mutant in the embodiment of the invention. Wherein the sequences and PAM sequences of the target sites EMX1-1, the off-target sites EMX1-1-OT1 and EMX1-1-OT2 are shown in table 1.

TABLE 1 target and off-target site target and PAM sequences

Wherein the thickened part is a base of which the off-target site is inconsistent with the target site.

ddPCR (Droplet Digital PCR, microdroplet digital PCR) was used to verify the efficiency of editing at the target site and off-target site.

For the sequences of the target sites EMX1-1, the off-target sites EMX1-1-OT1 and EMX1-1-OT2 shown in Table 1, primer sets were designed for amplifying a fragment of about 60-200bp long containing the target site or the off-target site, respectively, while Reference probes (containing FAM fluorescent modification groups) were designed for targeting the sequences at both ends of the sheared target site, and NHEJ probes (containing VIC fluorescent modification groups) were designed for targeting the sheared target site. The NHEJ probe can bind to the target site sequence when the target site is not edited, and can not bind to the target site any more when the target site is edited, so that the off-target condition can be judged by identifying the fluorescence of the VIC on the target sequence.

Wherein, the primer group, reference probe and NHEJ probe sequences of the target site EMX1-1, off-target site EMX1-1-OT1 and EMX1-1-OT2 are respectively as follows:

(1) Target site EMX1-1:

primer set sequence:

upstream primer F:5'-cggaggacaaagtacaaacgg-3' (SEQ ID NO. 10);

the downstream primer R:5'-gtcattggaggtgacatcgatg-3' (SEQ ID NO. 11).

The Reference probe sequences were: 5'-FAM-ccattggcctgcttcgtggcaatgcg-BHQ1-3' (SEQ ID NO. 12).

The NHEJ probe sequence was: 5'-VIC-cgagcagaagaagaag-MGB-3' (SEQ ID NO. 13).

(2) Off-target sites EMX1-1-OT1:

primer set sequence:

upstream primer F:5'-gctacctgtacatctgcacaag-3' (SEQ ID NO. 14);

the downstream primer R:5'-aagaaatgcccaatcattgatgc-3' (SEQ ID NO. 15).

The Reference probe sequences were: 5'-FAM-ctgtcttgccatgccataagcccctatt-BHQ1-3' (SEQ ID NO. 16).

The NHEJ probe sequence was: 5'-VIC-atgcctttcttcttc-MGB-3' (SEQ ID NO. 17).

(3) Off-target sites EMX1-1-OT2:

primer set sequence:

upstream primer F:5'-agcctctttctcaatgtgcttc-3' (SEQ ID NO. 18);

the downstream primer R:5'-agagtagatggttgggtagtgg-3' (SEQ ID NO. 19).

The Reference probe sequences were: 5'-FAM-ccatcacggcctttgcaaatagagccct-BHQ1-3' (SEQ ID NO. 20).

The NHEJ probe sequence was: 5'-VIC-ctaagcagaagaagaagag-MGB-3' (SEQ ID NO. 21).

Preparation of microdroplets in ddPCR reference QX200 ^TM Microdroplet digital PCR systemOr using other procedures conventional in the art.

The ddPCR reaction system is as follows:

TABLE 2 ddPCR reaction System

The reaction procedure is: pre-denaturation at 95℃for 10min; denaturation at 94℃for 30s; annealing at 50-65 ℃ for 1min; cycling for 40 times; 98 ℃ for 10min; maintained at 4 ℃.

Detection using a QX200TM microdroplet digital PCR system: the droplets are separated in sequence by a droplet analyzer, fluorescence signal detection is carried out one by one, and fluorescence signal values of each droplet in the FAM channel and the VIC channel are detected respectively. Droplets with fluorescent signal were positive and droplets without fluorescent signal were negative. The number and the ratio of positive droplets in each sample were recorded. And calculating editing efficiency according to the proportional relation.

The editing efficiency is calculated by using the NHEJ mutation rate, and the formula is as follows:

the results are shown in FIGS. 3-4 and Table 3.

FIGS. 2-3 are two-dimensional graphs of editing efficiency results of wild-type SpCas9 and mutant SpCas9 (K510A) at three sites of EMX1-1, EMX1-1-OT2, respectively. The blue dot in FIG. 2 is single FAM positive, the green dot is single VIC positive, the gray dot is FAM/VIC double negative, and the brown dot is FAM/VIC double positive. Further analysis found that wild-type SpCas9 had an edit rate of 53.31% for the target site EMX1-1, 2.51% for the off-target site EMX1-1-OT1, and 1.76% for the off-target site EMX1-1-OT 2. SpCas9 (K510A) mutant has an editing rate of 41.47% for the target site EMX1-1, 0.24% for the off-target site EMX1-1-OT1 and 0.08% for the off-target site EMX1-1-OT 2. Compared with the wild type SpCas9, the editing efficiency of the mutant SpCas9 (K510A) on the target site is basically the same as that of the wild type SpCas9 (more than 70%), and the editing rate on the off-target site is greatly reduced.

TABLE 3 editing efficiency of wild-type SpCas9 and mutant SpCas9 (K510A) at EMX1-1 target site and off-target site

vs. wild type, P <0.01, P <0.001.

Editing efficiency verification of SpCas9 (K510A) mutant at target site EMX1-1

Editing efficiency of SpCas9 (K510A) mutants was detected using T7E1 cleavage.

Primers were designed for the EMX1-1 site (720 bp amplification product length):

upstream primer F:5'-CTTCCAGAGCCTGCACTCCT-3' (SEQ ID NO. 22);

the downstream primer R:5'-AGGCTCTCCGAGGAGAAGGC-3' (SEQ ID NO. 23).

The primer is usedThe EMX1-1 gene sequence was amplified using Hot Start High-Fidelity 2X Master Mix premix (M0494, NEB). The amplified product was subjected to T7E1 cleavage (E3321, NEB). The results are shown in Table 4.

TABLE 4T 7E1 enzymatic cleavage assay results

Wild type, P <0.05.

The results showed that the cleavage rate of SpCas9 mutant (K510A) for the target site EMX1-1 was 72.0% of the cleavage rate of the wild-type (table 4), consistent with the results of ddPCR detection.

In summary, the SpCas9 (K510A) mutant in the embodiment of the present invention is designed by mutating the 510 th lysine residue of the wild SpCas9 to an alanine residue. The editing condition of the target sequence is detected through a T7E1 experiment, and ddPCR proves that under the condition that the cleavage efficiency of the SpCas9 (K510A) mutant to the target site is basically the same as that of the wild SpCas9, the editing efficiency of the off-target site can be obviously reduced, namely the off-target rate is obviously reduced.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.

SEQUENCE LISTING

<110> university of medical science in south China

<120> a CRISPR SpCas9 mutant and application thereof

<130>

<160> 23

<170> PatentIn version 3.5

<210> 1

<211> 1404

<212> PRT

<213> artificial sequence

<400> 1

Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val

1 5 10 15

Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe

20 25 30

Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile

35 40 45

Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu

50 55 60

Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys

65 70 75 80

Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser

85 90 95

Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys

100 105 110

His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr

115 120 125

His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp

130 135 140

Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His

145 150 155 160

Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro

165 170 175

Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr

180 185 190

Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala

195 200 205

Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn

210 215 220

Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn

225 230 235 240

Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe

245 250 255

Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp

260 265 270

Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp

275 280 285

Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp

290 295 300

Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser

305 310 315 320

Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys

325 330 335

Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe

340 345 350

Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser

355 360 365

Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp

370 375 380

Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg

385 390 395 400

Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu

405 410 415

Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe

420 425 430

Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile

435 440 445

Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp

450 455 460

Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu

465 470 475 480

Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr

485 490 495

Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser

500 505 510

Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys

515 520 525

Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln

530 535 540

Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr

545 550 555 560

Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp

565 570 575

Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly

580 585 590

Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp

595 600 605

Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr

610 615 620

Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala

625 630 635 640

His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr

645 650 655

Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp

660 665 670

Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe

675 680 685

Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe

690 695 700

Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu

705 710 715 720

His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly

725 730 735

Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly

740 745 750

Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln

755 760 765

Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile

770 775 780

Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro

785 790 795 800

Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu

805 810 815

Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg

820 825 830

Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys

835 840 845

Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg

850 855 860

Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys

865 870 875 880

Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys

885 890 895

Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp

900 905 910

Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr

915 920 925

Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp

930 935 940

Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser

945 950 955 960

Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg

965 970 975

Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val

980 985 990

Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe

995 1000 1005

Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala

1010 1015 1020

Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe

1025 1030 1035

Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala

1040 1045 1050

Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu

1055 1060 1065

Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val

1070 1075 1080

Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr

1085 1090 1095

Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys

1100 1105 1110

Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro

1115 1120 1125

Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val

1130 1135 1140

Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys

1145 1150 1155

Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser

1160 1165 1170

Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys

1175 1180 1185

Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu

1190 1195 1200

Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly

1205 1210 1215

Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val

1220 1225 1230

Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser

1235 1240 1245

Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys

1250 1255 1260

His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys

1265 1270 1275

Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala

1280 1285 1290

Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn

1295 1300 1305

Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala

1310 1315 1320

Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser

1325 1330 1335

Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr

1340 1345 1350

Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp

1355 1360 1365

Gly Ser Pro Lys Lys Lys Arg Lys Val Ser Ser Asp Tyr Lys Asp

1370 1375 1380

His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp Tyr Lys Asp Asp

1385 1390 1395

Asp Asp Lys Ala Ala Gly

1400

<210> 2

<211> 1404

<212> PRT

<213> artificial sequence

<400> 2

Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val

1 5 10 15

Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe

20 25 30

Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile

35 40 45

Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu

50 55 60

Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys

65 70 75 80

Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser

85 90 95

Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys

100 105 110

His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr

115 120 125

His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp

130 135 140

Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His

145 150 155 160

Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro

165 170 175

Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr

180 185 190

Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala

195 200 205

Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn

210 215 220

Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn

225 230 235 240

Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe

245 250 255

Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp

260 265 270

Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp

275 280 285

Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp

290 295 300

Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser

305 310 315 320

Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys

325 330 335

Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe

340 345 350

Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser

355 360 365

Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp

370 375 380

Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg

385 390 395 400

Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu

405 410 415

Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe

420 425 430

Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile

435 440 445

Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp

450 455 460

Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu

465 470 475 480

Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr

485 490 495

Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Ala His Ser

500 505 510

Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys

515 520 525

Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln

530 535 540

Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr

545 550 555 560

Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp

565 570 575

Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly

580 585 590

Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp

595 600 605

Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr

610 615 620

Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala

625 630 635 640

His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr

645 650 655

Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp

660 665 670

Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe

675 680 685

Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe

690 695 700

Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu

705 710 715 720

His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly

725 730 735

Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly

740 745 750

Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln

755 760 765

Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile

770 775 780

Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro

785 790 795 800

Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu

805 810 815

Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg

820 825 830

Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys

835 840 845

Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg

850 855 860

Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys

865 870 875 880

Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys

885 890 895

Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp

900 905 910

Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr

915 920 925

Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp

930 935 940

Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser

945 950 955 960

Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg

965 970 975

Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val

980 985 990

Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe

995 1000 1005

Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala

1010 1015 1020

Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe

1025 1030 1035

Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala

1040 1045 1050

Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu

1055 1060 1065

Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val

1070 1075 1080

Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr

1085 1090 1095

Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys

1100 1105 1110

Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro

1115 1120 1125

Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val

1130 1135 1140

Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys

1145 1150 1155

Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser

1160 1165 1170

Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys

1175 1180 1185

Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu

1190 1195 1200

Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly

1205 1210 1215

Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val

1220 1225 1230

Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser

1235 1240 1245

Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys

1250 1255 1260

His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys

1265 1270 1275

Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala

1280 1285 1290

Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn

1295 1300 1305

Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala

1310 1315 1320

Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser

1325 1330 1335

Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr

1340 1345 1350

Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp

1355 1360 1365

Gly Ser Pro Lys Lys Lys Arg Lys Val Ser Ser Asp Tyr Lys Asp

1370 1375 1380

His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp Tyr Lys Asp Asp

1385 1390 1395

Asp Asp Lys Ala Ala Gly

1400

<210> 3

<211> 20

<212> DNA

<213> artificial sequence

<400> 3

gagtccgagc agaagaagaa 20

<210> 4

<211> 7614

<212> DNA

<213> artificial sequence

<400> 4

cggatcggga gatcgatctc ccgatcccct agggtcgact ctcagtacaa tctgctctga 60

tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg 120

cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct 180

gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca 240

ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata 300

tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga 360

cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt 420

ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt 480

gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca 540

ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt 600

catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt 660

tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca 720

ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg 780

cggtaggcgt gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat 840

ccgctagaga tccgcggccg ctaatacgac tcactatagg gagagccgcc accatggata 900

aaaagtattc tattggttta gacatcggca ctaattccgt tggatgggct gtcataaccg 960

atgaatacaa agtaccttca aagaaattta aggtgttggg gaacacagac cgtcattcga 1020

ttaaaaagaa tcttatcggt gccctcctat tcgatagtgg cgaaacggca gaggcgactc 1080

gcctgaaacg aaccgctcgg agaaggtata cacgtcgcaa gaaccgaata tgttacttac 1140

aagaaatttt tagcaatgag atggccaaag ttgacgattc tttctttcac cgtttggaag 1200

agtccttcct tgtcgaagag gacaagaaac atgaacggca ccccatcttt ggaaacatag 1260

tagatgaggt ggcatatcat gaaaagtacc caacgattta tcacctcaga aaaaagctag 1320

ttgactcaac tgataaagcg gacctgaggt taatctactt ggctcttgcc catatgataa 1380

agttccgtgg gcactttctc attgagggtg atctaaatcc ggacaactcg gatgtcgaca 1440

aactgttcat ccagttagta caaacctata atcagttgtt tgaagagaac cctataaatg 1500

caagtggcgt ggatgcgaag gctattctta gcgcccgcct ctctaaatcc cgacggctag 1560

aaaacctgat cgcacaatta cccggagaga agaaaaatgg gttgttcggt aaccttatag 1620

cgctctcact aggcctgaca ccaaatttta agtcgaactt cgacttagct gaagatgcca 1680

aattgcagct tagtaaggac acgtacgatg acgatctcga caatctactg gcacaaattg 1740

gagatcagta tgcggactta tttttggctg ccaaaaacct tagcgatgca atcctcctat 1800

ctgacatact gagagttaat actgagatta ccaaggcgcc gttatccgct tcaatgatca 1860

aaaggtacga tgaacatcac caagacttga cacttctcaa ggccctagtc cgtcagcaac 1920

tgcctgagaa atataaggaa atattctttg atcagtcgaa aaacgggtac gcaggttata 1980

ttgacggcgg agcgagtcaa gaggaattct acaagtttat caaacccata ttagagaaga 2040

tggatgggac ggaagagttg cttgtaaaac tcaatcgcga agatctactg cgaaagcagc 2100

ggactttcga caacggtagc attccacatc aaatccactt aggcgaattg catgctatac 2160

ttagaaggca ggaggatttt tatccgttcc tcaaagacaa tcgtgaaaag attgagaaaa 2220

tcctaacctt tcgcatacct tactatgtgg gacccctggc ccgagggaac tctcggttcg 2280

catggatgac aagaaagtcc gaagaaacga ttactccatg gaattttgag gaagttgtcg 2340

ataaaggtgc gtcagctcaa tcgttcatcg agaggatgac caactttgac aagaatttac 2400

cgaacgaaaa agtattgcct aagcacagtt tactttacga gtatttcaca gtgtacaatg 2460

aactcacgaa agttaagtat gtcactgagg gcatgcgtaa acccgccttt ctaagcggag 2520

aacagaagaa agcaatagta gatctgttat tcaagaccaa ccgcaaagtg acagttaagc 2580

aattgaaaga ggactacttt aagaaaattg aatgcttcga ttctgtcgag atctccgggg 2640

tagaagatcg atttaatgcg tcacttggta cgtatcatga cctcctaaag ataattaaag 2700

ataaggactt cctggataac gaagagaatg aagatatctt agaagatata gtgttgactc 2760

ttaccctctt tgaagatcgg gaaatgattg aggaaagact aaaaacatac gctcacctgt 2820

tcgacgataa ggttatgaaa cagttaaaga ggcgtcgcta tacgggctgg ggacgattgt 2880

cgcggaaact tatcaacggg ataagagaca agcaaagtgg taaaactatt ctcgattttc 2940

taaagagcga cggcttcgcc aataggaact ttatgcagct gatccatgat gactctttaa 3000

ccttcaaaga ggatatacaa aaggcacagg tttccggaca aggggactca ttgcacgaac 3060

atattgcgaa tcttgctggt tcgccagcca tcaaaaaggg catactccag acagtcaaag 3120

tagtggatga gctagttaag gtcatgggac gtcacaaacc ggaaaacatt gtaatcgaga 3180

tggcacgcga aaatcaaacg actcagaagg ggcaaaaaaa cagtcgagag cggatgaaga 3240

gaatagaaga gggtattaaa gaactgggca gccagatctt aaaggagcat cctgtggaaa 3300

atacccaatt gcagaacgag aaactttacc tctattacct acaaaatgga agggacatgt 3360

atgttgatca ggaactggac ataaaccgtt tatctgatta cgacgtcgat cacattgtac 3420

cccaatcctt tttgaaggac gattcaatcg acaataaagt gcttacacgc tcggataaga 3480

accgagggaa aagtgacaat gttccaagcg aggaagtcgt aaagaaaatg aagaactatt 3540

ggcggcagct cctaaatgcg aaactgataa cgcaaagaaa gttcgataac ttaactaaag 3600

ctgagagggg tggcttgtct gaacttgaca aggccggatt tattaaacgt cagctcgtgg 3660

aaacccgcca aatcacaaag catgttgcac agatactaga ttcccgaatg aatacgaaat 3720

acgacgagaa cgataagctg attcgggaag tcaaagtaat cactttaaag tcaaaattgg 3780

tgtcggactt cagaaaggat tttcaattct ataaagttag ggagataaat aactaccacc 3840

atgcgcacga cgcttatctt aatgccgtcg tagggaccgc actcattaag aaatacccga 3900

agctagaaag tgagtttgtg tatggtgatt acaaagttta tgacgtccgt aagatgatcg 3960

cgaaaagcga acaggagata ggcaaggcta cagccaaata cttcttttat tctaacatta 4020

tgaatttctt taagacggaa atcactctgg caaacggaga gatacgcaaa cgacctttaa 4080

ttgaaaccaa tggggagaca ggtgaaatcg tatgggataa gggccgggac ttcgcgacgg 4140

tgagaaaagt tttgtccatg ccccaagtca acatagtaaa gaaaactgag gtgcagaccg 4200

gagggttttc aaaggaatcg attcttccaa aaaggaatag tgataagctc atcgctcgta 4260

aaaaggactg ggacccgaaa aagtacggtg gcttcgatag ccctacagtt gcctattctg 4320

tcctagtagt ggcaaaagtt gagaagggaa aatccaagaa actgaagtca gtcaaagaat 4380

tattggggat aacgattatg gagcgctcgt cttttgaaaa gaaccccatc gacttccttg 4440

aggcgaaagg ttacaaggaa gtaaaaaagg atctcataat taaactacca aagtatagtc 4500

tgtttgagtt agaaaatggc cgaaaacgga tgttggctag cgccggagag cttcaaaagg 4560

ggaacgaact cgcactaccg tctaaatacg tgaatttcct gtatttagcg tcccattacg 4620

agaagttgaa aggttcacct gaagataacg aacagaagca actttttgtt gagcagcaca 4680

aacattatct cgacgaaatc atagagcaaa tttcggaatt cagtaagaga gtcatcctag 4740

ctgatgccaa tctggacaaa gtattaagcg catacaacaa gcacagggat aaacccatac 4800

gtgagcaggc ggaaaatatt atccatttgt ttactcttac caacctcggc gctccagccg 4860

cattcaagta ttttgacaca acgatagatc gcaaacgata cacttctacc aaggaggtgc 4920

tagacgcgac actgattcac caatccatca cgggattata tgaaactcgg atagatttgt 4980

cacagcttgg gggtgacgga tcccccaaga agaagaggaa agtctcgagc gactacaaag 5040

accatgacgg tgattataaa gatcatgaca tcgattacaa ggatgacgat gacaaggctg 5100

caggatgacc ggtcatcatc accatcacca ttgagtttaa acccgctgat cagcctcgac 5160

tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct 5220

ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct 5280

gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg 5340

ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag 5400

aaccagctgg ggctcgatac cgtcgacctc tagctagagc ttggcgtaat catggtcata 5460

gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag 5520

cataaagtgt aaagcctagg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg 5580

ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca 5640

acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc 5700

gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg 5760

gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa 5820

ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga 5880

cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag 5940

ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct 6000

taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg 6060

ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc 6120

ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt 6180

aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta 6240

tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac 6300

agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc 6360

ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat 6420

tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc 6480

tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt 6540

cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta 6600

aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct 6660

atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg 6720

cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga 6780

tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt 6840

atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt 6900

taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt 6960

tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat 7020

gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc 7080

cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc 7140

cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat 7200

gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag 7260

aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt 7320

accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc 7380

ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa 7440

gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg 7500

aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa 7560

taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcga 7614

<210> 5

<211> 7614

<212> DNA

<213> artificial sequence

<400> 5

cggatcggga gatcgatctc ccgatcccct agggtcgact ctcagtacaa tctgctctga 60

tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg 120

cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct 180

gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca 240

ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata 300

tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga 360

cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt 420

ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt 480

gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca 540

ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt 600

catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt 660

tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca 720

ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg 780

cggtaggcgt gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat 840

ccgctagaga tccgcggccg ctaatacgac tcactatagg gagagccgcc accatggata 900

aaaagtattc tattggttta gacatcggca ctaattccgt tggatgggct gtcataaccg 960

atgaatacaa agtaccttca aagaaattta aggtgttggg gaacacagac cgtcattcga 1020

ttaaaaagaa tcttatcggt gccctcctat tcgatagtgg cgaaacggca gaggcgactc 1080

gcctgaaacg aaccgctcgg agaaggtata cacgtcgcaa gaaccgaata tgttacttac 1140

aagaaatttt tagcaatgag atggccaaag ttgacgattc tttctttcac cgtttggaag 1200

agtccttcct tgtcgaagag gacaagaaac atgaacggca ccccatcttt ggaaacatag 1260

tagatgaggt ggcatatcat gaaaagtacc caacgattta tcacctcaga aaaaagctag 1320

ttgactcaac tgataaagcg gacctgaggt taatctactt ggctcttgcc catatgataa 1380

agttccgtgg gcactttctc attgagggtg atctaaatcc ggacaactcg gatgtcgaca 1440

aactgttcat ccagttagta caaacctata atcagttgtt tgaagagaac cctataaatg 1500

caagtggcgt ggatgcgaag gctattctta gcgcccgcct ctctaaatcc cgacggctag 1560

aaaacctgat cgcacaatta cccggagaga agaaaaatgg gttgttcggt aaccttatag 1620

cgctctcact aggcctgaca ccaaatttta agtcgaactt cgacttagct gaagatgcca 1680

aattgcagct tagtaaggac acgtacgatg acgatctcga caatctactg gcacaaattg 1740

gagatcagta tgcggactta tttttggctg ccaaaaacct tagcgatgca atcctcctat 1800

ctgacatact gagagttaat actgagatta ccaaggcgcc gttatccgct tcaatgatca 1860

aaaggtacga tgaacatcac caagacttga cacttctcaa ggccctagtc cgtcagcaac 1920

tgcctgagaa atataaggaa atattctttg atcagtcgaa aaacgggtac gcaggttata 1980

ttgacggcgg agcgagtcaa gaggaattct acaagtttat caaacccata ttagagaaga 2040

tggatgggac ggaagagttg cttgtaaaac tcaatcgcga agatctactg cgaaagcagc 2100

ggactttcga caacggtagc attccacatc aaatccactt aggcgaattg catgctatac 2160

ttagaaggca ggaggatttt tatccgttcc tcaaagacaa tcgtgaaaag attgagaaaa 2220

tcctaacctt tcgcatacct tactatgtgg gacccctggc ccgagggaac tctcggttcg 2280

catggatgac aagaaagtcc gaagaaacga ttactccatg gaattttgag gaagttgtcg 2340

ataaaggtgc gtcagctcaa tcgttcatcg agaggatgac caactttgac aagaatttac 2400

cgaacgaaaa agtattgcct gcccacagtt tactttacga gtatttcaca gtgtacaatg 2460

aactcacgaa agttaagtat gtcactgagg gcatgcgtaa acccgccttt ctaagcggag 2520

aacagaagaa agcaatagta gatctgttat tcaagaccaa ccgcaaagtg acagttaagc 2580

aattgaaaga ggactacttt aagaaaattg aatgcttcga ttctgtcgag atctccgggg 2640

tagaagatcg atttaatgcg tcacttggta cgtatcatga cctcctaaag ataattaaag 2700

ataaggactt cctggataac gaagagaatg aagatatctt agaagatata gtgttgactc 2760

ttaccctctt tgaagatcgg gaaatgattg aggaaagact aaaaacatac gctcacctgt 2820

tcgacgataa ggttatgaaa cagttaaaga ggcgtcgcta tacgggctgg ggacgattgt 2880

cgcggaaact tatcaacggg ataagagaca agcaaagtgg taaaactatt ctcgattttc 2940

taaagagcga cggcttcgcc aataggaact ttatgcagct gatccatgat gactctttaa 3000

ccttcaaaga ggatatacaa aaggcacagg tttccggaca aggggactca ttgcacgaac 3060

atattgcgaa tcttgctggt tcgccagcca tcaaaaaggg catactccag acagtcaaag 3120

tagtggatga gctagttaag gtcatgggac gtcacaaacc ggaaaacatt gtaatcgaga 3180

tggcacgcga aaatcaaacg actcagaagg ggcaaaaaaa cagtcgagag cggatgaaga 3240

gaatagaaga gggtattaaa gaactgggca gccagatctt aaaggagcat cctgtggaaa 3300

atacccaatt gcagaacgag aaactttacc tctattacct acaaaatgga agggacatgt 3360

atgttgatca ggaactggac ataaaccgtt tatctgatta cgacgtcgat cacattgtac 3420

cccaatcctt tttgaaggac gattcaatcg acaataaagt gcttacacgc tcggataaga 3480

accgagggaa aagtgacaat gttccaagcg aggaagtcgt aaagaaaatg aagaactatt 3540

ggcggcagct cctaaatgcg aaactgataa cgcaaagaaa gttcgataac ttaactaaag 3600

ctgagagggg tggcttgtct gaacttgaca aggccggatt tattaaacgt cagctcgtgg 3660

aaacccgcca aatcacaaag catgttgcac agatactaga ttcccgaatg aatacgaaat 3720

acgacgagaa cgataagctg attcgggaag tcaaagtaat cactttaaag tcaaaattgg 3780

tgtcggactt cagaaaggat tttcaattct ataaagttag ggagataaat aactaccacc 3840

atgcgcacga cgcttatctt aatgccgtcg tagggaccgc actcattaag aaatacccga 3900

agctagaaag tgagtttgtg tatggtgatt acaaagttta tgacgtccgt aagatgatcg 3960

cgaaaagcga acaggagata ggcaaggcta cagccaaata cttcttttat tctaacatta 4020

tgaatttctt taagacggaa atcactctgg caaacggaga gatacgcaaa cgacctttaa 4080

ttgaaaccaa tggggagaca ggtgaaatcg tatgggataa gggccgggac ttcgcgacgg 4140

tgagaaaagt tttgtccatg ccccaagtca acatagtaaa gaaaactgag gtgcagaccg 4200

gagggttttc aaaggaatcg attcttccaa aaaggaatag tgataagctc atcgctcgta 4260

aaaaggactg ggacccgaaa aagtacggtg gcttcgatag ccctacagtt gcctattctg 4320

tcctagtagt ggcaaaagtt gagaagggaa aatccaagaa actgaagtca gtcaaagaat 4380

tattggggat aacgattatg gagcgctcgt cttttgaaaa gaaccccatc gacttccttg 4440

aggcgaaagg ttacaaggaa gtaaaaaagg atctcataat taaactacca aagtatagtc 4500

tgtttgagtt agaaaatggc cgaaaacgga tgttggctag cgccggagag cttcaaaagg 4560

ggaacgaact cgcactaccg tctaaatacg tgaatttcct gtatttagcg tcccattacg 4620

agaagttgaa aggttcacct gaagataacg aacagaagca actttttgtt gagcagcaca 4680

aacattatct cgacgaaatc atagagcaaa tttcggaatt cagtaagaga gtcatcctag 4740

ctgatgccaa tctggacaaa gtattaagcg catacaacaa gcacagggat aaacccatac 4800

gtgagcaggc ggaaaatatt atccatttgt ttactcttac caacctcggc gctccagccg 4860

cattcaagta ttttgacaca acgatagatc gcaaacgata cacttctacc aaggaggtgc 4920

tagacgcgac actgattcac caatccatca cgggattata tgaaactcgg atagatttgt 4980

cacagcttgg gggtgacgga tcccccaaga agaagaggaa agtctcgagc gactacaaag 5040

accatgacgg tgattataaa gatcatgaca tcgattacaa ggatgacgat gacaaggctg 5100

caggatgacc ggtcatcatc accatcacca ttgagtttaa acccgctgat cagcctcgac 5160

tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct 5220

ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct 5280

gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg 5340

ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag 5400

aaccagctgg ggctcgatac cgtcgacctc tagctagagc ttggcgtaat catggtcata 5460

gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag 5520

cataaagtgt aaagcctagg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg 5580

ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca 5640

acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc 5700

gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg 5760

gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa 5820

ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga 5880

cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag 5940

ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct 6000

taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg 6060

ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc 6120

ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt 6180

aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta 6240

tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac 6300

agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc 6360

ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat 6420

tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc 6480

tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt 6540

cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta 6600

aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct 6660

atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg 6720

cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga 6780

tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt 6840

atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt 6900

taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt 6960

tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat 7020

gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc 7080

cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc 7140

cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat 7200

gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag 7260

aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt 7320

accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc 7380

ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa 7440

gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg 7500

aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa 7560

taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcga 7614

<210> 6

<211> 8408

<212> DNA

<213> artificial sequence

<400> 6

aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca 60

tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga 120

tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt 180

gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg 240

gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc 300

tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 360

taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg 420

aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt 480

gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg 540

actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga 600

attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta 660

aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta 720

gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga 780

tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg 840

atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt 900

aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga 960

caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc 1020

acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc 1080

tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct 1140

gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag 1200

ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca 1260

ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg 1320

ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa 1380

atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa 1440

ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 1500

acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa 1560

ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620

agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt 1680

tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg 1740

tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcgcta 1800

gcttttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata 1860

atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt 1920

actagtgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt 1980

agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga 2040

cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac 2100

tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg 2160

aaaggacgaa acaccggagt ccgagcagaa gaagaagttt tagagctaga aatagcaagt 2220

taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttgt 2280

ctagaggtac cgaattccaa ctttgtatag aaaagttggg gttgcgcctt ttccaaggca 2340

gccctgggtt tgcgcaggga cgcggctgct ctgggcgtgg ttccgggaaa cgcagcggcg 2400

ccgaccctgg gtctcgcaca ttcttcacgt ccgttcgcag cgtcacccgg atcttcgccg 2460

ctacccttgt gggccccccg gcgacgcttc ctgctccgcc cctaagtcgg gaaggttcct 2520

tgcggttcgc ggcgtgccgg acgtgacaaa cggaagccgc acgtctcact agtaccctcg 2580

cagacggaca gcgccaggga gcaatggcag cgcgccgacc gcgatgggct gtggccaata 2640

gcggctgctc agcagggcgc gccgagagca gcggccggga aggggcggtg cgggaggcgg 2700

ggtgtggggc ggtagtgtgg gccctgttcc tgcccgcgcg gtgttccgca ttctgcaagc 2760

ctccggagcg cacgtcggca gtcggctccc tcgttgaccg aatcaccgac ctctctcccc 2820

aggcaagttt gtacaaaaaa gcaggctgcc accatggtga gcaagggcga ggagctgttc 2880

accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc 2940

gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc 3000

accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg 3060

cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg 3120

cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc 3180

cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc 3240

gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac 3300

aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc 3360

cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc 3420

ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc 3480

aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg 3540

atcactctcg gcatggacga gctgtacaag ggctccggag agggcagggg aagtcttcta 3600

acatgcgggg acgtggagga aaatcccggc cccatgaccg agtacaagcc cacggtgcgc 3660

ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac 3720

taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg 3780

caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac 3840

ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc 3900

gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg 3960

gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc 4020

gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag 4080

gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc 4140

ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc 4200

acctggtgca tgacccgcaa gcccggtgcc tgaacccagc tttcttgtac aaagtggtgg 4260

tacccgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact 4320

atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg 4380

cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg 4440

aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa 4500

cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc 4560

ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg 4620

ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaagctgacg tcctttccat 4680

ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt 4740

cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc 4800

cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc 4860

ggctttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa 4920

ggggggactg gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac 4980

tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc 5040

actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt 5100

gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag 5160

cagtagtagt tcatgtcatc ttattattca gtatttataa cttgcaaaga aatgaatatc 5220

agagagtgag aggaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat 5280

cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact 5340

catcaatgta tcttatcatg tctggctcta gctatcccgc ccctaactcc gcccatcccg 5400

cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt 5460

tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt 5520

tttggaggcc tagggacgta cccaattcgc cctatagtga gtcgtattac gcgcgctcac 5580

tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc 5640

ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc 5700

cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa 5760

gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc 5820

ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag 5880

ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca 5940

aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc 6000

gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa 6060

cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct 6120

attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa 6180

cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 6240

tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca 6300

ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt 6360

ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga 6420

tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa 6480

gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct 6540

gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat 6600

acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga 6660

tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc 6720

caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat 6780

gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa 6840

cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac 6900

tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa 6960

agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc 7020

tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc 7080

ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag 7140

acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta 7200

ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa 7260

gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc 7320

gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat 7380

ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga 7440

gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt 7500

tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata 7560

cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac 7620

cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg 7680

ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg 7740

tgagctatga gaaagcgcca cgcttcccga agagagaaag gcggacaggt atccggtaag 7800

cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct 7860

ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc 7920

aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt 7980

ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg 8040

tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga 8100

gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg 8160

gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg 8220

caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct 8280

tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta 8340

tgaccatgat tacgccaagc gcgcaattaa ccctcactaa agggaacaaa agctggagct 8400

gcaagctt 8408

<210> 7

<211> 20

<212> DNA

<213> artificial sequence

<400> 7

gagtccgagc agaagaagaa 20

<210> 8

<211> 20

<212> DNA

<213> artificial sequence

<400> 8

gagttagagc agaagaagaa 20

<210> 9

<211> 20

<212> DNA

<213> artificial sequence

<400> 9

gagtctaagc agaagaagaa 20

<210> 10

<211> 21

<212> DNA

<213> artificial sequence

<400> 10

cggaggacaa agtacaaacg g 21

<210> 11

<211> 22

<212> DNA

<213> artificial sequence

<400> 11

gtcattggag gtgacatcga tg 22

<210> 12

<211> 26

<212> DNA

<213> artificial sequence

<400> 12

ccattggcct gcttcgtggc aatgcg 26

<210> 13

<211> 16

<212> DNA

<213> artificial sequence

<400> 13

cgagcagaag aagaag 16

<210> 14

<211> 22

<212> DNA

<213> artificial sequence

<400> 14

gctacctgta catctgcaca ag 22

<210> 15

<211> 23

<212> DNA

<213> artificial sequence

<400> 15

aagaaatgcc caatcattga tgc 23

<210> 16

<211> 28

<212> DNA

<213> artificial sequence

<400> 16

ctgtcttgcc atgccataag cccctatt 28

<210> 17

<211> 15

<212> DNA

<213> artificial sequence

<400> 17

atgcctttct tcttc 15

<210> 18

<211> 22

<212> DNA

<213> artificial sequence

<400> 18

agcctctttc tcaatgtgct tc 22

<210> 19

<211> 22

<212> DNA

<213> artificial sequence

<400> 19

agagtagatg gttgggtagt gg 22

<210> 20

<211> 28

<212> DNA

<213> artificial sequence

<400> 20

ccatcacggc ctttgcaaat agagccct 28

<210> 21

<211> 19

<212> DNA

<213> artificial sequence

<400> 21

ctaagcagaa gaagaagag 19

<210> 22

<211> 20

<212> DNA

<213> artificial sequence

<400> 22

cttccagagc ctgcactcct 20

<210> 23

<211> 20

<212> DNA

<213> artificial sequence

<400> 23

aggctctccg aggagaaggc 20

Claims

1. The SpCas9 mutant is characterized in that the amino acid sequence of the SpCas9 mutant is shown as SEQ ID NO. 2.

2. A composition comprising the SpCas9 mutant of claim 1.

3. A polynucleotide encoding the SpCas9 mutant of claim 1.

4. A guide-polynucleotide/Cas complex, characterized in that the guide-polynucleotide/Cas complex comprises a guide-polynucleotide and the SpCas9 mutant of claim 1;

wherein the nucleotide sequence of the guide-polynucleotide is shown as SEQ ID NO. 3.

5. A recombinant vector, recombinant bacterium or cell comprising the polynucleotide of claim 3; the cells are derived from non-human animals, bacteria, fungi, insects or yeast.

6. A method of modifying a target site in a cell genome for non-diagnostic and therapeutic purposes, comprising: introducing the guide-polynucleotide/Cas complex of claim 4 into a cell.

7. Use of the SpCas9 mutant of claim 1 in the preparation of a gene editing product.