CN112522264B - CRISPR/Cas9 system causing congenital deafness and application thereof in preparation of model pig nuclear donor cells - Google Patents

Abstract

The invention discloses a CRISPR/Cas9 system causing congenital deafness and application thereof in preparing model pig nuclear donor cells. A gRNA expression vector for porcine TMC1 gene, expressing SEQ ID NO: 25. A CRISPR/Cas9 system for pig TMC1 gene editing comprises a Cas9 expression vector and the gRNA expression vector, wherein the Cas9 expression vector is a plasmid full sequence shown as SEQ ID NO. 2. According to the invention, four gRNAs are designed aiming at the pig TMC1 gene, and the high-efficiency gRNAs are screened from the gRNAs and then the pre-set targets are knocked out, so that the workload of later monoclonal cell identification and screening can be effectively reduced, and the PCR product sequencing can be directly used for detecting the gene editing efficiency. The efficient expression vector of Cas9 modified by the invention is adopted for gene editing, and the editing efficiency is improved by more than 100% compared with the original vector.

Description

CRISPR/Cas9 system causing congenital deafness and application thereof in preparation of model pig nuclear donor cells

Technical Field

The invention belongs to the technical field of biology, relates to a CRISPR/Cas9 system causing congenital deafness and application thereof in preparing model pig nuclear donor cells, and in particular relates to the CRISPR/Cas9 system and application thereof in preparing TMC1 gene mutation congenital genetic deafness clone pig nuclear donor cells.

Background

Congenital deafness (congenital deafness) refers to deafness caused by abnormalities or genetic factors in pregnancy, childbirth, and is mostly sensorineural deafness. Congenital deafness can be divided into two major categories, hereditary and non-hereditary. 43 syndrome type deafness genes and 99 non-syndrome type deafness genes have been found. Wherein, a plurality of genes can cause autosomal dominant hereditary non-syndrome type deafness and autosomal recessive hereditary non-syndrome type deafness simultaneously, and TMC1 is one of them. TMC1 was first reported in 2002 to have multiple mutations that lead to dominant (DFNA 36) and recessive (DFNB 7/11) hereditary hearing loss. TMC1 is a necessary component for detecting acoustic wave stimulation by auditory hair cells in the inner ear, but its physiological role and deafness causing mechanism are still unclear at present, so development of an animal model for congenital genetic deafness caused by TMC1 mutation is urgently needed to study its pathogenic mechanism and therapeutic regimen. Pigs are main meat supply animals for a long time, are easy to breed and raise on a large scale, have lower requirements on ethical morals, animal protection and the like, have similar body size and physiological functions to human beings, and are ideal human disease model animals.

Gene editing is a biotechnology that has been greatly developed in recent years, and includes editing means from gene editing based on homologous recombination to ZFN, TALEN, CRISPR/Cas9 based on nuclease, wherein CRISPR/Cas9 technology is currently the most advanced gene editing technology. Currently, gene editing techniques are increasingly applied to the production of animal models.

Disclosure of Invention

The invention aims at overcoming the defects of the prior art and providing an sgRNA target and a gRNA for TMC1 gene editing.

It is another object of the present invention to provide a CRISPR/Cas9 system for porcine TMC1 gene editing.

It is a further object of the present invention to provide for the use of the CRISPR/Cas9 system.

The aim of the invention can be achieved by the following technical scheme:

an sgRNA target for TMC1 gene editing, the sequence of which is shown in SEQ ID NO: 21.

A gRNA for TMC1 gene editing, the sequence of which is shown in SEQ ID NO: 25.

A gRNA expression vector for pig TMC1 gene uses pKG-U6gRNA with the complete sequence shown in SEQ ID NO.3 as a vector skeleton to express the gRNA for TMC1 gene editing.

Preferably, the expression vector of the present invention is a vector comprising the sequence of SEQ ID NO:32 and SEQ ID NO:33, and a double-stranded DNA molecule having a cohesive end obtained by annealing the single-stranded DNA shown in FIG. 33 is inserted into the BbsI restriction enzyme site of the vector backbone pKG-U6 gRNA.

A CRISPR/Cas9 system for pig TMC1 gene editing, comprising a Cas9 expression vector and a gRNA expression vector for pig TMC1 gene according to the invention.

As one preferable choice of the invention, the Cas9 expression vector is a pU6gRNA-eEF1a-mNLS-hSpCas9-EGFP-PURO vector with a plasmid full sequence shown as SEQ ID NO. 2.

To increase the gene editing capacity of the Cas9 Plasmid, we modified pU6gRNA-eEF1a-mNLS-hSpCas9-EGFP-PURO (abbreviated as granule pKG-GE 3) on the basis of the vector pX330-U6-Chimeric_BB-CBh-hSpCas9 (abbreviated as PX 330) purchased from adedge (Plasmid #42230,from Zhang Feng lab). The map of PX330 is shown in fig. 1, modified as follows:

1) Removing redundant invalid sequences in the gRNA skeleton of the original vector;

2) Modifying a promoter: the original promoter (chicken beta-actin promoter) is modified into EF1a promoter with higher expression activity, so that the protein expression capacity of the Cas9 gene is increased;

3) Increasing the nuclear localization signal: adding a nuclear localization signal coding sequence (NLS) at the N end and the C end of the Cas9, and increasing the nuclear localization capability of the Cas 9;

4) Adding double screening markers: the original vector does not have any screening mark, is not beneficial to screening and enrichment of positive transformed cells, and P2A-EGFP-T2A-PURO is inserted into the C end of Cas9, so that the fluorescence and resistance screening capability of the vector are endowed;

5) Inserting WPRE, 3' LTR and other sequences for regulating gene expression: the WPRE, 3' LTR and other sequences are inserted into the gene frame at last, so that the protein translation capacity of the Cas9 gene can be enhanced.

The modified vector pU6gRNA-eEF1a-mNLS-hSpCas9-EGFP-PURO (called pKG-GE3 for short) is shown in figure 2, and the plasmid has the full sequence shown in SEQ ID NO:2 is shown in the figure; the main elements of pKG-GE3 are:

1) gRNA expression element: u6gRNA scaffold;

2) Promoter: EF1a promoter and CMV enhancer;

3) Cas9 gene comprising multiple NLSs: cas9 gene containing N-terminal and C-terminal polynuclear localization signals (NLS);

4) Screening marker genes: fluorescent and resistant double selectable marker element P2A-EGFP-T2A-PURO;

5) Element for enhancing translation: WPRE and 3' LTR enhance the translation efficiency of Cas9 and selectable marker genes;

6) Transcription termination signal: bGH polyA signal;

7) A carrier skeleton: including Amp resistance elements and ori replicons, and the like.

The plasmid pKG-GE3 has a specific fusion gene; the specific fusion gene codes for a specific fusion protein;

the specific fusion protein sequentially comprises the following elements from the N end to the C end: two Nuclear Localization Signals (NLS), cas9 protein, two nuclear localization signals, self-cleaving polypeptide P2A, fluorescent reporter protein, self-cleaving polypeptide T2A, resistance selection marker protein;

in the plasmid pKG-GE3, the EF1a promoter is used for promoting the expression of the specific fusion gene;

in plasmid pKG-GE3, the specific fusion gene has downstream a WPRE sequence element, a 3' LTR sequence element and a bGH poly (A) signal sequence element.

The plasmid pKG-GE3 has the following elements in this order: CMV enhancer, EF1a promoter, the specific fusion gene, WPRE sequence element, 3' LTR sequence element, bGH poly (A) signal sequence element.

In the specific fusion protein, two nuclear localization signals at the upstream of the Cas9 protein are SV40 nuclear localization signals, and two nuclear localization signals at the downstream of the Cas9 protein are nucleoplasin nuclear localization signals.

In the specific fusion protein, the fluorescent reporter protein can be EGFP protein.

In the specific fusion protein, the resistance screening marker protein can be a Puromycin protein.

The amino acid sequence of the self-cleaving polypeptide P2A is "ATNFSLLKQAGDVEENPGP" (the cleavage site where self-cleavage occurs is between the first amino acid residue and the second amino acid residue from the C-terminus).

The amino acid sequence of the self-cleaving polypeptide T2A is "EGRGSLLTCGDVEENPGP" (the cleavage site where self-cleavage occurs is between the first amino acid residue and the second amino acid residue from the C-terminus).

Specific fusion genes are specifically shown as SEQ ID NO:2 from nucleotide numbers 911-6706.

CMV enhancer as set forth in SEQ ID NO:2 from nucleotide 395 to 680.

The EF1a promoter is shown in SEQ ID NO:2 from nucleotide 682 to nucleotide 890.

WPRE sequence element is shown as SEQ ID NO:2 from nucleotide 6722 to nucleotide 7310.

The 3' LTR sequence element is shown in SEQ ID NO:2 from nucleotide 7382 to nucleotide 7615.

The bGH poly (A) signal sequence element is shown as SEQ ID NO:2 from nucleotide 7647 to nucleotide 7871.

As a further preferred aspect of the invention, the molar ratio of the gRNA expression vector to Cas9 expression vector is 1-3:1, more preferably 3:1.

The CRISPR/Cas9 system is applied to construction of congenital genetic deafness clone pig nuclear donor cells.

A recombinant porcine fibroblast cell characterized by being obtained by the verification of the CRISPR/Cas9 system co-transfected porcine primary fibroblast cell of any one of claims 5-7.

The recombinant cell is applied to construction of TMC1 gene knockout cloned pigs; preferably in constructing cloned pigs with TMC1 gene knocked out congenital genetic deafness.

Compared with the prior art, the invention has at least the following beneficial effects:

(1) The subject (pig) of the invention has better applicability than other animals (rats, mice, primates).

Rodents such as rats and mice have great differences from humans in terms of body type, organ size, physiology, pathology and the like, and cannot truly simulate normal physiological and pathological states of humans. Studies have shown that more than 95% of drugs that are validated in mice are ineffective in human clinical trials. In the case of large animals, primates are animals that are closest to human relatives, but are small in size, late in sexual maturity (mating begins at 6-7 years old), and single animals, the population expansion rate is extremely slow, and the raising cost is high. In addition, primate cloning is inefficient, difficult and costly.

The pig is an animal which has the closest relationship with human except primate, and has the similar size, weight, organ size and the like to human, and is very similar to human in terms of anatomy, physiology, nutrition metabolism, disease pathogenesis and the like. Meanwhile, the pigs are early in sexual maturity (4-6 months), have high fertility and have more piglets, and can form a larger group within 2-3 years. In addition, the cloning technology of pigs is very mature, and the cloning and feeding cost is much lower than that of primates; the pig is taken as a meat animal for a long time, and the pig is taken as a disease model animal, so that the requirements on animal protection, ethics and the like are low.

(2) According to the invention, four gRNAs are designed aiming at the pig TMC1 gene, and the high-efficiency gRNAs are screened from the gRNAs and then the pre-set targets are knocked out, so that the workload of later-stage identification and screening can be effectively reduced, and the PCR product sequencing can be directly used for detecting the gene editing efficiency.

(3) The efficient expression vector of Cas9 modified by the invention is adopted for gene editing, and the editing efficiency is improved by more than 100% compared with the original vector.

(4) The efficient expression vector of the Cas9 modified by the invention is adopted for gene editing, and the genotype [ homozygous mutation (including homozygous mutation of the same variation of the double alleles and homozygous mutation of different variations of the double alleles), heterozygous mutation or wild type ], of the obtained cells can be analyzed according to the sequencing result of the target gene PCR product, wherein the probability of obtaining the homozygous mutation is 30% -50%, which is greatly superior to the probability (lower than 5%) of obtaining the homozygous mutation in a model preparation method (namely fertilized ovum injection gene editing material) using embryo injection technology.

(5) The homozygous mutant monoclonal cell strain obtained by the invention is used for cloning somatic cell nuclear transfer animals, so that cloned pigs containing target gene homozygous mutation can be directly obtained, and the homozygous mutation can be stably inherited.

The method of microinjection of gene editing material into fertilized ovum and embryo transplantation adopted in mouse model production is not suitable for large animal (such as pig) model production with long gestation period because the probability of directly obtaining homozygous mutant offspring is very low (less than 5%), and the offspring need to be hybridized and bred. Therefore, the method for editing and screening the positive editing monoclonal cells in vitro by using the primary cells with high technical difficulty and high challenge is adopted, and the corresponding disease model pigs are directly obtained by somatic cell nuclear transfer animal cloning technology in the later period, so that the manufacturing period of the model pigs can be greatly shortened, and the manpower, material resources and financial resources are saved.

The invention lays a solid foundation for obtaining the congenital deafness pig model by a gene editing means, is helpful for researching and revealing the pathogenesis of the congenital deafness caused by TMC1 mutation, can be further used for researching drug screening, drug effect detection, disease pathology, gene therapy, cell therapy and the like, can provide effective experimental data for further clinical application, and further provides a powerful experimental means for successfully treating the congenital deafness caused by TMC1 mutation. The invention has great application value for research and development of congenital genetic deafness drugs and revealing pathogenesis of the disease.

Drawings

FIG. 1 is a schematic diagram of the structure of plasmid pX330.

FIG. 2 is a schematic diagram of the structure of plasmid pKG-GE3.

FIG. 3 is a schematic representation of the structure of pU6gRNACas9 vector.

FIG. 4 is a structural map of pU6gRNA-eEF1a Cas9 vector.

FIG. 5 is a pU6gRNA-eEF1a Cas9+nNLS vector map.

FIG. 6 is a schematic diagram of the structure of plasmid pKG-U6 gRNA.

FIG. 7 is a schematic representation of the insertion of a DNA molecule of about 20bp (target sequence binding region for transcription to form gRNA) into plasmid pKG-U6 gRNA.

FIG. 8 shows the sequencing results when the plasmid proportioning was optimized.

FIG. 9 shows the sequencing results when the effects of plasmid pX330 and plasmid pKG-GE3 are compared.

FIG. 10 is an electrophoresis chart of example 3 after PCR amplification using 18 pig genomic DNAs as templates.

FIG. 11 is a plot of sequencing peaks in step four of example 3.

FIG. 12 is an electrophoresis chart of target gene PCR products of the monoclonal cells obtained in example 4.

FIG. 13 shows the results of forward sequencing of monoclonal cells numbered TMC1-27 in comparison to wild type.

FIG. 14 shows the results of forward sequencing of monoclonal cells numbered TMC1-11 in comparison to wild type.

FIG. 15 shows the results of forward sequencing of monoclonal cells numbered TMC1-3 in comparison to wild type.

FIG. 16 is a result of forward sequencing of monoclonal cells numbered TMC1-14 in comparison to wild type.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. The recombinant plasmids constructed in the examples were all subjected to sequencing verification. Complete culture solution (% by volume): 15% fetal bovine serum (Gibco) +83% DMEM medium (Gibco) +1% Penicillin-Streptomycin (Gibco) +1% HEPES (Solarbio). Cell culture conditions: 37 ℃,5% CO ₂ 、5％O ₂ Is a constant temperature incubator.

A method of preparing porcine primary fibroblasts: (1) taking 0.5g of pig ear tissue, removing hair, soaking in 75% alcohol for 30-40s, washing with PBS buffer solution containing 5% (volume ratio) Penicillin-Streptomycin (Gibco) for 5 times, and washing with PBS buffer solution for one time; (2) shearing the tissue with scissors, digesting with 5mL 1% collagenase solution (Sigma) at 37deg.C for 1h, centrifuging 500g for 5min, and discarding the supernatant; (3) the pellet was resuspended in 1mL of complete medium, then plated into 10-diameter cell culture dishes containing 10mL of complete medium and capped with 0.2% gelatin (VWR) and cultured to about 60% of the cell growth bottom; (4) after step (3) is completed, cells are digested and harvested with trypsin and then resuspended in complete medium.

Example 1 preparation of plasmids

1.1 preparation of plasmid pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO (abbreviated as plasmid pKG-GE 3)

Original plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9 (abbreviated as plasmid pX330, the sequence is shown as SEQ ID NO: 1. The schematic structure of plasmid pX330 is shown in FIG. 1.SEQ ID NO:1, nucleotides 440-725 constitute CMV enhancer, nucleotides 727-1208 constitute chicken beta-actin promoter, nucleotides 1304-1324 encode SV40 Nuclear Localization Signal (NLS), nucleotides 1325-5449 encode Cas9 protein, and nucleotides 5450-5497 encode nucleotoplastmin Nuclear Localization Signal (NLS).

Plasmid pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO, abbreviated as plasmid pKG-GE3, the nucleotide is shown as SEQ ID NO: 2. The construction method comprises the following steps:

(1) Removal of superfluous and ineffective sequences from gRNA backbone

Plasmid pX330 was digested with BbsI and XbaI, the vector fragment (about 8313 bp) was recovered, the insert 175bp (SEQ ID NO: 4) was synthesized by a multi-fragment recombination method, and the recovered vector fragment was recombined to give pU6gRNACas9 vector (FIG. 3).

(2) Modified promoter and enhancer

For the constructed pU6gRNACas9 vector, the promoter (chicken beta-actin promoter) and enhancer sequence (CMV enhancer) were removed by XbaI and AgeI endonucleases, the linear vector sequence was recovered to about 7650bp, 554bp of sequence containing CMV enhancer and EF1a promoter (SEQ ID NO: 5) was synthesized by multi-fragment recombination, and pU6gRNA-eEF1a Cas9 vector was obtained by recombination with the digested vector pU6gRNACas9 (FIG. 4).

(3) N-terminal increased NLS sequence of Cas9 gene

And (3) carrying out enzyme digestion on the constructed vector pU6gRNA-eEF1a Cas9 by using AgeI and BglII, recovering 7786bp vector sequence, supplementing the sequence added with NLS to enzyme digestion sites, namely synthesizing 447bp Cas9 coding sequence (SEQ ID NO: 6) comprising 2 nuclear localization signals and partial excision by utilizing a multi-fragment recombination method, and recombining to obtain the pU6gRNA-eEF1a Cas9+nNLS vector (figure 5).

(4) Adding NLS, P2A-EGFP-T2A-PURO and WPRE-3' LTR-bGH polyA signals into C end of Cas9 gene

The constructed vector is named pU6gRNA-eEF1a Cas9+nNLS, fseI and SbfI are used for enzyme digestion, 7781bp of vector sequence is recovered, 2727bp of sequence comprising NLS-P2A-EGFP-T2A-PURO-WPRE-3' LTR-bGH polyA signals (SEQ ID NO: 7) is synthesized by utilizing a multi-fragment recombination method, and the sequence is recombined with a vector fragment to obtain the vector pU6gRNA-eEF1a-mNLS-hSpCas9-EGFP-PURO, which is called pKG-GE3 for short, and the plasmid map is shown as figure 2 and the nucleotide sequence (SEQ ID NO: 2).

SEQ ID NO:2, nucleotides 395 to 680 comprising the CMV enhancer, nucleotides 682 to 890 comprising the EF1a promoter, nucleotides 986 to 1006 comprising the Nuclear Localization Signal (NLS), nucleotides 1016 to 1036 comprising the Nuclear Localization Signal (NLS), nucleotides 1037 to 5161 comprising the Cas9 protein, nucleotides 5162 to 5209 comprising the Nuclear Localization Signal (NLS), nucleotides 5219 to 5266 comprising the Nuclear Localization Signal (NLS), nucleotides 5276 to 5332 comprising the self-cleaving polypeptide P2A (the amino acid sequence of the self-cleaving polypeptide P2A is "ATNFSLLKQAGDVEENPGP", the cleavage site for the self-cleaving is between the first amino acid residue and the second amino acid residue from the C-terminus), nucleotide numbers 5333-6046 encode EGFP protein, nucleotide numbers 6056-6109 encode self-cleaving polypeptide T2A (the amino acid sequence of self-cleaving polypeptide T2A is EGRGSLLTCGDVEENPGP, the cleavage site where self-cleavage occurs is between the first amino acid residue and the second amino acid residue from the C-terminus), nucleotide numbers 6110-6703 encode Puromycin protein (called Puro protein for short), nucleotide numbers 6722-7310 constitute WPRE sequence element, nucleotide numbers 7382-7615 constitute 3' LTR sequence element, and nucleotide numbers 7647-7871 constitute bGH poly (A) signal sequence element. SEQ ID NO:2, 911-6706 form a fusion gene, expressing a fusion protein. Due to the presence of self-cleaving polypeptide P2A and self-cleaving polypeptide T2A, the fusion protein spontaneously forms three proteins: proteins with Cas9 protein, proteins with EGFP protein, and proteins with Puro protein.

Compared with plasmid pX330, plasmid pKG-GE3 was mainly modified as follows: (1) removing residual gRNA backbone sequences (GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTTT), reducing interference; (2) the original chicken beta-actin promoter is modified into an EF1a promoter with higher expression activity, so that the protein expression capacity of the Cas9 gene is increased; (3) adding nuclear localization signal coding genes (NLS) at the upstream and downstream of the Cas9 gene, and increasing the nuclear localization capability of the Cas9 protein; (4) the original plasmid has no eukaryotic cell screening mark, is not beneficial to screening and enrichment of positive transformed cells, and is sequentially inserted with P2A-EGFP-T2A-PURO coding genes at the downstream of Cas9 genes, so that the carrier fluorescence and eukaryotic cell resistance screening capability are endowed; (5) the insertion of the WPRE element and the 3' ltr sequence element enhances the protein translation capacity of the Cas9 gene.

1.2 construction of pKG-U6gRNA vector

The pUC57 vector is obtained by connecting a pKG-U6gRNA insertion sequence (a DNA fragment containing a U6 promoter, a BbsI restriction enzyme site and an sgRNA framework sequence, and the sequence is shown as SEQ ID NO: 8) through an EcoRV restriction enzyme site, and reversely inserting the DNA fragment into the pUC57 vector to obtain a complete sequence (SEQ ID NO: 3) of the pKG-U6gRNA vector, wherein the complete sequence is shown as SEQ ID NO:3, nucleotides 2280 to 2539 constitute the hU6 promoter and nucleotides 2558 to 2637 are used for transcription to form the gRNA backbone. When in use, a DNA molecule (target sequence binding region for transcription to form gRNA) of about 20bp is inserted into plasmid pKG-U6gRNA to form a recombinant plasmid, the schematic diagram is shown in FIG. 4, and the recombinant plasmid is transcribed in cells to obtain gRNA. The constructed pKG-U6gRNA vector is shown in FIG. 6.

Example 2 comparison of the effects of plasmid pX330 and plasmid pKG-GE3

Selecting a high-efficiency gRNA target located in the RAG1 gene:

target for RAG1-gRNA 4: 5'-AGTTATGGCAGAACTCAGTG-3' (SEQ ID NO: 9).

Primers used to amplify and detect fragments containing the target were as follows:

RAG1-nF126：5’-CCCCATCCAAAGTTTTTAAAGGA-3’(SEQ ID NO：10)；

RAG1-nR525：5’-TGTGGCAGATGTCACAGTTTAGG-3’(SEQ ID NO：11)

primary swine fibroblasts were prepared from Jiang Xiang swine (female, blood group AO).

1. Preparation of recombinant plasmids

Plasmid pKG-U6gRNA was taken and digested with restriction enzyme BbsI, and the vector backbone (about 3kb linear fragment) was recovered. RAG1-4S and RAG1-4A were synthesized separately, and then mixed and annealed to give a double-stranded DNA molecule having cohesive ends. The double-stranded DNA molecule having a cohesive end and the vector backbone were ligated to obtain plasmid pKG-U6gRNA (RAG 1-gRNA 4).

RAG1-4S：5’-caccgAGTTATGGCAGAACTCAGTG-3’(SEQ ID NO：12)；

RAG1-4A：5’-aaacCACTGAGTTCTGCCATAACTc-3’(SEQ ID NO：13)。

RAG1-4S and RAG1-4A are single stranded DNA molecules.

2. Plasmid proportioning optimization

A first group: the plasmid pKG-U6gRNA (RAG 1-gRNA 4) and plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Proportioning: about 20 ten thousand porcine primary fibroblasts: 0.44. Mu.g plasmid pKG-U6gRNA (RAG 1-gRNA 4): 1.56. Mu.g of plasmid pKG-GE3. Namely, the molar ratio of the plasmid pKG-U6gRNA (RAG 1-gRNA 4) to the plasmid pKG-GE3 is as follows: 1:1.

second group: the plasmid pKG-U6gRNA (RAG 1-gRNA 4) and plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Proportioning: about 20 ten thousand porcine primary fibroblasts: 0.72. Mu.g plasmid pKG-U6gRNA (RAG 1-gRNA 4): 1.28. Mu.g of plasmid pKG-GE3. Namely, the molar ratio of the plasmid pKG-U6gRNA (RAG 1-gRNA 4) to the plasmid pKG-GE3 is as follows: 2:1.

third group: the plasmid pKG-U6gRNA (RAG 1-gRNA 4) and plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Proportioning: about 20 ten thousand porcine primary fibroblasts: 0.92. Mu.g of plasmid pKG-U6gRNA (RAG 1-gRNA 4): 1.08 μg of plasmid pKG-GE3. Namely, the molar ratio of the plasmid pKG-U6gRNA (RAG 1-gRNA 4) to the plasmid pKG-GE3 is as follows: 3:1.

fourth group: plasmid pKG-U6gRNA (RAG 1-gRNA 4) was transfected into porcine primary fibroblasts. Proportioning: about 20 ten thousand porcine primary fibroblasts: mu.g of plasmid pKG-U6gRNA (RAG 1-gRNA 4).

Co-transfection was performed by electric shock transfection using a mammalian nuclear transfection kit (Neon kit, thermofiser) and a Neon TM transfection system electrotransfection apparatus (parameters set to 1450V, 10ms, 3 pulses).

2. After the step 1 is completed, the culture is carried out for 16 to 18 hours by adopting the complete culture solution, and then the culture is carried out by replacing the new complete culture solution. The total incubation time was 48 hours.

3. After step 2 is completed, cells are digested and collected by trypsin, genomic DNA is extracted, PCR amplification is performed by using a primer pair consisting of RAG1-nF126 and RAG1-nR525, and then electrophoresis is performed.

The band of interest was recovered after electrophoresis and sequenced, and the sequencing results are shown in FIG. 8.

The efficiency of editing of different targets was obtained by analyzing the sequencing peak plots using the synthetic ICE tool. The gene editing efficiency of the first group to the third group was 9%, 53%, 66% in this order. The fourth group did not undergo gene editing. The results showed that the third set of edits were most efficient, determining the optimum amount of single gRNA plasmid to Cas9 plasmid as a molar ratio of 3:1, the actual amount of plasmid was 0.92. Mu.g: 1.08 μg. 3. Comparison of the effects of plasmid pX330 and plasmid pKG-GE3

1. Co-transfection

RAG1-B group: plasmid pKG-U6gRNA (RAG 1-gRNA 4) was transfected into porcine primary fibroblasts. Proportioning: about 20 ten thousand porcine primary fibroblasts: 0.92. Mu.g of plasmid pKG-U6gRNA (RAG 1-gRNA 4).

RAG1-330 group: plasmid pKG-U6gRNA (RAG 1-gRNA 4) and plasmid pX330 were co-transfected into porcine primary fibroblasts. Proportioning: about 20 ten thousand porcine primary fibroblasts: 0.92. Mu.g of plasmid pKG-U6gRNA (RAG 1-gRNA 4): 1.08 μg of plasmid pX330.

RAG1-KG group: the plasmid pKG-U6gRNA (RAG 1-gRNA 4) and plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Proportioning: about 20 ten thousand porcine primary fibroblasts: 0.92. Mu.g of plasmid pKG-U6gRNA (RAG 1-gRNA 4): 1.08 μg of plasmid pKG-GE3.

Co-transfection was performed by electric shock transfection using a mammalian nuclear transfection kit (Neon kit, thermofiser) and a Neon TM transfection system electrotransfection apparatus (parameters set to 1450V, 10ms, 3 pulses).

2. After the step 1 is completed, the culture is carried out for 16 to 18 hours by adopting the complete culture solution, and then the culture is carried out by replacing the new complete culture solution. The total incubation time was 48 hours.

3. After step 2 is completed, cells are digested and collected by trypsin, genomic DNA is extracted, PCR amplification is carried out by using a primer pair consisting of RAG1-nF126 and RAG1-nR525, and the products are sequenced.

The efficiency of editing of different targets was obtained by analyzing the sequencing peak plots using the synthetic ICE tool. No gene editing occurred in RAG1-B groups. The editing efficiency of RAG1-330 groups and RAG1-KG groups is 28% and 68% in sequence. Exemplary peak diagrams of sequencing results are shown in FIG. 9. The results show that the use of plasmid pKG-GE3 results in a significant increase in gene editing efficiency compared to the use of plasmid pX330.

Example 3 target screening for TMC1 Gene knockout

Porcine TMC1 gene information: encoding ransmembrane channel like protein; is located on chromosome 1 of pig;

GeneID is 100516949,Sus scrofa. The protein encoded by the porcine TMC1 gene is shown as GENBANK ACCESSION No. XP_020920511.1 (linear MAM 13-MAY-2017). In the genome DNA, the pig TMC1 gene has 27 exons, wherein the 12 th exon and the 500bp sequences above and below the 12 th exon are shown in SEQ ID NO:14, the encoded protein fragment is shown as SEQ ID NO: 15.

1. TMC1 gene knockout preset target point and adjacent genome sequence conservation analysis

18 junior from Jiang fragrant pigs, of which 10 females (named 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, respectively) and 8 males (named A, B, C, D, E, F, G, H, respectively) were female.

PCR amplification was performed using 18 pig genomic DNAs as templates, respectively, and primer pairs (the target sequences of the primer pairs include exon 12 of pig TMC1 gene), followed by electrophoresis. And (3) recovering PCR amplification products, sequencing, and comparing the sequencing results with TMC1 gene sequences in a public database for analysis. Based on the results of the alignment, primers for detecting the mutation (the primers themselves avoid possible mutation sites) were designed. The primers designed for mutation detection were: TMC1-E12-F/TMC1-E12-R. The electrophoresis pattern of 18 pig genomic DNA obtained by PCR amplification using the primer pair consisting of TMC1-E12-F/TMC1-E12-R is shown in FIG. 10.

TMC1-E12-F：5’-GCATCCCACCTTACACTAGGAA-3’(SEQ ID NO：16)；

TMC1-E12-R：5’-CAGAGCGATGAGGGGATGAT-3’(SEQ ID NO：17)。

2. Screening target

A plurality of targets are initially screened by screening NGG (avoiding possible mutation sites), and 4 targets are further screened from the targets through preliminary experiments.

The 4 targets were as follows:

sgRNA _TMC1-E12-g1 target point: 5'-TTGGACAGAACATCCCCAGG-3’(SEQ ID NO：18)；

sgRNA _TMC1-E12-g2 Target point: 5'-ACATGGTCATGTCCCTCCTG-3' (SEQ ID NO: 19);

sgRNA _TMC1-E12-g3 target point: 5'-TGGACAGAACATCCCCAGGA-3' (SEQ ID NO: 20);

sgRNA _TMC1-E12-g4 target point: 5'-GTGTTGGACAGAACATCCCC-3' (SEQ ID NO: 21).

3. Preparation of recombinant plasmids

Plasmid pKG-U6gRNA was taken and digested with restriction enzyme BbsI, and the vector backbone (about 3kb linear fragment) was recovered.

TMC1-E12-gRNA1-S and TMC1-E12-gRNA1-A were synthesized separately, and then mixed and annealed to give double-stranded DNA molecules with cohesive ends. The double-stranded DNA molecule having a cohesive end was ligated to the vector backbone to give plasmid pKG-U6gRNA (TMC 1-E12-g 1). Plasmid pKG-U6gRNA (TMC 1-E12-g 1) expresses the sequence of SEQ ID NO:22, sgrnas as shown _TMC1-E12-g1 。

SEQ ID NO：22：

UUGGACAGAACAUCCCCAGGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu

TMC1-E12-gRNA2-S and TMC1-E12-gRNA2-A were synthesized separately, and then mixed and annealed to give double-stranded DNA molecules with cohesive ends. The double-stranded DNA molecule having a cohesive end was ligated to the vector backbone to give plasmid pKG-U6gRNA (TMC 1-E12-g 2). Plasmid pKG-U6gRNA (TMC 1-E12-g 2) expresses the sequence of SEQ ID NO:23, sgRNA shown in FIG. 23 _TMC1-E12-g2 。

SEQ ID NO：23：

ACAUGGUCAUGUCCCUCCUGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu

TMC1-E12-gRNA3-S and TMC1-E12-gRNA3-A were synthesized separately, and then mixed and annealed to give double-stranded DNA molecules with cohesive ends. The double-stranded DNA molecule having a cohesive end was ligated to the vector backbone to give plasmid pKG-U6gRNA (TMC 1-E12-g 3). Plasmid pKG-U6gRNA (TMC 1-E12-g 3) expresses the sequence of SEQ ID NO:24, sgRNA shown in FIG. 24 _TMC1-E12-g3 。

SEQ ID NO：24：

UGGACAGAACAUCCCCAGGAguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu

TMC1-E12-gRNA4-S and TMC1-E12-gRNA4-A were synthesized separately, and then mixed and annealed to give double-stranded DNA molecules with cohesive ends. The double-stranded DNA molecule having a cohesive end was ligated to the vector backbone to give plasmid pKG-U6gRNA (TMC 1-E12-g 4). Plasmid pKG-U6gRNA (TMC 1-E12-g 4) expresses the sequence of SEQ ID NO:25, sgRNA as indicated in FIG. 25 _TMC1-E12-g4 。

SEQ ID NO：25：

GUGUUGGACAGAACAUCCCCguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu

TMC1-E12-gRNA1-S：5’-caccgTTGGACAGAACATCCCCAGG-3’(SEQ ID NO：26)；

TMC1-E12-gRNA1-A：5’-aaacCCTGGGGATGTTCTGTCCAAc-3’(SEQ ID NO：27)；

TMC1-E12-gRNA2-S：5’-caccgACATGGTCATGTCCCTCCTG-3’(SEQ ID NO：28)；

TMC1-E12-gRNA2-A：5’-aaacCAGGAGGGACATGACCATGTc-3’(SEQ ID NO：29)；

TMC1-E12-gRNA3-S：5’-caccgTGGACAGAACATCCCCAGGA-3’(SEQ ID NO：30)；

TMC1-E12-gRNA3-A：5’-aaacTCCTGGGGATGTTCTGTCCAc-3’(SEQ ID NO：31)；

TMC1-E12-gRNA4-S：5’-caccGTGTTGGACAGAACATCCCC-3’(SEQ ID NO：32)；

TMC1-E12-gRNA4-A：5’-aaacGGGGATGTTCTGTCCAACAC-3’(SEQ ID NO：33)。

TMC1-E12-gRNA1-S, TMC1-E12-gRNA1-A, TMC1-E12-gRNA2-S, TMC1-E12-gRNA2-A, TMC1-E12-gRNA3-S, TMC1-E12-gRNA3-A, TMC1-E12-gRNA4-S, TMC1-E12-gRNA4-A are single stranded DNA molecules.

4. Editing efficiency comparison of different targets

Porcine primary fibroblasts were prepared from ear tissue of a junior river-flavored pig (female, blood group AO).

1. Co-transfection

A first group: plasmid pKG-U6gRNA (TMC 1-E12-g 1) and plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Proportioning: about 20 ten thousand porcine primary fibroblasts: 0.92. Mu.g of plasmid pKG-U6gRNA (TMC 1-E12-g 1): 1.08 μg of plasmid pKG-GE3.

Second group: plasmid pKG-U6gRNA (TMC 1-E12-g 2) and plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Proportioning: about 20 ten thousand porcine primary fibroblasts: 0.92. Mu.g of plasmid pKG-U6gRNA (TMC 1-E12-g 2): 1.08 μg of plasmid pKG-GE3.

Third group: plasmid pKG-U6gRNA (TMC 1-E12-g 3) and plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Proportioning: about 20 ten thousand porcine primary fibroblasts: 0.92. Mu.g of plasmid pKG-U6gRNA (TMC 1-E12-g 3): 1.08 μg of plasmid pKG-GE3.

Fourth group: plasmid pKG-U6gRNA (TMC 1-E12-g 4) and plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Proportioning: about 20 ten thousand porcine primary fibroblasts: 0.92. Mu.g of plasmid pKG-U6gRNA (TMC 1-E12-g 4): 1.08 μg of plasmid pKG-GE3.

Fifth group: pig primary fibroblasts were not subjected to any transfection procedure.

Co-transfection was performed by electric shock transfection using a mammalian nuclear transfection kit (Neon kit, thermofiser) and a Neon TM transfection system electrotransfection apparatus (parameters set to 1450V, 10ms, 3 pulses).

2. After the step 1 is completed, the culture is carried out for 16 to 18 hours by adopting the complete culture solution, and then the culture is carried out by replacing the new complete culture solution. The total incubation time was 48 hours.

3. After the completion of step 2, cells were digested with trypsin and collected, then lysed and genomic DNA was extracted, PCR amplified using a primer pair consisting of TMC1-E12-F and TMC1-E12-R, and then electrophoresed. The target fragment was recovered and sequenced, and the sequencing peaks are shown in FIG. 11. Analysis of the sequencing peak map using the synthetic ICE tool resulted in gene editing efficiencies for different targets. The gene editing efficiency of the first group to the fourth group was 29%, 35%, 39%, 45% in this order. The fifth group did not undergo gene editing. The results showed that the fourth set of editing was most efficient, sgRNA _TMC1-E12-g4 The target point of (2) is the optimal target point.

Example 4 preparation of TMC1 Gene-editing monoclonal cells

Porcine primary fibroblasts were prepared from ear tissue of a junior river-flavored pig (female, blood group AO).

1. Co-transfection

Plasmid pKG-U6gRNA (TMC 1-E12-g 4) and plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Proportioning: about 20 ten thousand porcine primary fibroblasts: 0.92. Mu.g of plasmid pKG-U6gRNA (TMC 1-E12-g 4): 1.08 μg of plasmid pKG-GE3.

Co-transfection was performed by electric shock transfection using a mammalian nuclear transfection kit (Neon kit, thermofiser) and a Neon TM transfection system electrotransfection apparatus (parameters set to 1450V, 10ms, 3 pulses).

2. After the step 1 is completed, the culture is carried out for 16 to 18 hours by adopting the complete culture solution, and then the culture is carried out by replacing the new complete culture solution. The total incubation time was 48 hours.

3. After step 2 was completed, the cells were digested with trypsin and collected, washed with complete broth, resuspended with complete broth, and then each single cell was individually picked into different wells of a 96-well plate (1 cell per well, 100 μl of complete broth per well) and cultured for 2 weeks (new complete broth was changed every 2-3 days).

4. After completion of step 3, cells were digested with trypsin and collected (about 2/3 of the resulting cells per well were inoculated into 6-well plates with complete culture medium, and the remaining 1/3 were collected in 1.5mL centrifuge tubes for subsequent genotyping detection).

5. The 6-well plate of step 4 was used to culture cells to 80% confluence, trypsinized and the cells were collected and frozen using cell frozen stock (90% complete medium+10% dmso, volume ratio).

6. Taking the centrifuge tube in the step 4, taking cells, extracting genome DNA, performing PCR amplification by adopting a primer pair consisting of TMC1-E12-F and TMC1-E12-R, and then performing electrophoresis. Porcine primary fibroblasts were used as wild-type controls. The electrophoresis pattern is shown in FIG. 12. The lane numbers in fig. 12 are consistent with the cell numbers in table 1.

7. After step 6 is completed, the PCR amplification product is recovered and sequenced.

The sequencing result of the primary fibroblast of the pig is only one, and the genotype of the primary fibroblast is wild type. If there are two kinds of sequencing results of a single clone cell, one kind is consistent with the sequencing results of the primary fibroblast of the pig, the other kind is mutated (mutation comprises deletion, insertion or substitution of one or more nucleotides) compared with the sequencing results of the primary fibroblast of the pig, the genotype of the single clone cell is heterozygous mutant; if the sequencing result of a certain monoclonal cell is two types, compared with the sequencing result of a swine primary fibroblast, mutation (mutation comprises deletion, insertion or replacement of one or more nucleotides), the genotype of the monoclonal cell is homozygotic mutation type with different variants of double alleles; if the sequencing result of a monoclonal cell is one and a mutation (mutation includes deletion, insertion or substitution of one or more nucleotides) is generated compared with the sequencing result of a swine primary fibroblast, the genotype of the monoclonal cell is a homozygotic mutant type of the same variation of the double alleles; if the sequencing result of a certain monoclonal cell is one and is consistent with the sequencing result of a primary fibroblast of a pig, the genotype of the monoclonal cell is wild type.

The results are shown in Table 1. The genotypes of the monoclonal cells numbered 11, 35, 36, 37, 38, 43, 48 are homozygotic mutants of the same variation of the double alleles. The genotypes of the monoclonal cells numbered 6, 10, 12, 13, 14, 15, 16, 20, 29, 31, 32, 44, 47 are homozygotic mutants of different variants of the double allele. The genotypes of the monoclonal cells numbered 3, 8, 19, 23 are heterozygous mutants. The monoclonal cells numbered 2, 4, 5, 7, 21, 22, 25, 26, 30, 33, 34, 39, 41, 42, 46 all showed complex sets of peaks, and therefore, an effective sequence could not be obtained, and the genotype and specific form could not be determined, but it could be judged that gene editing occurred. The ratio of the obtained gene editing monoclonal cells was 39/47.

Exemplary sequencing alignment results are shown in FIGS. 13-16. FIG. 13 is the result of forward sequencing of monoclonal cells numbered TMC1-27 as compared to wild type, as judged wild type. FIG. 14 is a result of forward sequencing of monoclonal cells numbered TMC1-11 aligned with the wild type, and a homozygous mutant version of the same variation of the biallelic was determined. FIG. 15 is a result of forward sequencing of monoclonal cells numbered TMC1-3 aligned with wild type, judged as heterozygous mutant. FIG. 16 is a result of forward sequencing of monoclonal cells numbered TMC1-14 aligned with the wild type, and a homozygous mutant version with different variants of the biallelic were determined.

TABLE 1

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Sequence listing

<110> Nanjing Kidney Gene engineering Co., ltd

<120> a CRISPR/Cas9 system causing congenital deafness and application thereof in preparing model pig nuclear donor cells

<160> 33

<170> SIPOSequenceListing 1.0

<210> 1

<211> 8484

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60

ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120

aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180

atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240

cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag 300

gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct 360

agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac 420

aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 480

ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc 540

aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 600

caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt 660

acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 720

ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac 780

ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg 840

ggggggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc 900

agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata 960

aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct 1020

ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga 1080

gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg taagggttta 1140

agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg cctgaaatca 1200

ctttttttca ggttggaccg gtgccaccat ggactataag gaccacgacg gagactacaa 1260

ggatcatgat attgattaca aagacgatga cgataagatg gccccaaaga agaagcggaa 1320

ggtcggtatc cacggagtcc cagcagccga caagaagtac agcatcggcc tggacatcgg 1380

caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt 1440

caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct 1500

gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata 1560

caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa 1620

ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa 1680

gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta 1740

ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg 1800

gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg 1860

cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta 1920

caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct 1980

gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga 2040

gaagaagaat ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt 2100

caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga 2160

cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc 2220

cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat 2280

caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct 2340

gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt 2400

cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt 2460

ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa 2520

gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca 2580

ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt 2640

cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt 2700

gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac 2760

catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat 2820

cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag 2880

cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga 2940

gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct 3000

gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat 3060

cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg 3120

cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa 3180

cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat 3240

cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa 3300

gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga 3360

caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa 3420

cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca 3480

ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc 3540

cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg 3600

ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa 3660

gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg 3720

cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta 3780

cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg 3840

gctgtccgac tacgatgtgg accatatcgt gcctcagagc tttctgaagg acgactccat 3900

cgacaacaag gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc 3960

cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat 4020

tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga 4080

taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc 4140

acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga 4200

agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt 4260

ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt 4320

cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga 4380

ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc 4440

taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct 4500

ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat 4560

cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt 4620

gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc 4680

caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg 4740

cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg 4800

caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag 4860

cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa 4920

ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag 4980

aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata 5040

tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa 5100

tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca 5160

gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc 5220

cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct 5280

gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga 5340

ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat 5400

caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca aaaggccggc 5460

ggccacgaaa aaggccggcc aggcaaaaaa gaaaaagtaa gaattcctag agctcgctga 5520

tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct 5580

tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca 5640

tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag 5700

ggggaggatt gggaagagaa tagcaggcat gctggggagc ggccgcagga acccctagtg 5760

atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag 5820

gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc 5880

ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc 5940

atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg 6000

tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt 6060

tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc 6120

tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg 6180

gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg 6240

agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct 6300

cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg 6360

agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat 6420

ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc 6480

caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag 6540

ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg 6600

cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg 6660

tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 6720

ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 6780

aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct 6840

tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 6900

atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 6960

agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 7020

tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca 7080

tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 7140

atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 7200

ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 7260

tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 7320

acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa 7380

ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 7440

aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 7500

ctggagccgg tgagcgtgga agccgcggta tcattgcagc actggggcca gatggtaagc 7560

cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 7620

gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 7680

actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 7740

agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 7800

cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 7860

tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 7920

agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 7980

ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 8040

acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 8100

ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 8160

gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 8220

gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 8280

gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 8340

tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 8400

caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 8460

tttgctggcc ttttgctcac atgt 8484

<210> 2

<211> 10476

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60

ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120

aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180

atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240

cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag 300

gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttc tagcgcgtgc 360

gccaattctg cagacaaatg gctctagagg tacccgttac ataacttacg gtaaatggcc 420

cgcctggctg accgcccaac gacccccgcc cattgacgtc aatagtaacg ccaataggga 480

ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 540

aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 600

ggcattgtgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 660

tagtcatcgc tattaccatg ggggcagagc gcacatcgcc cacagtcccc gagaagttgg 720

ggggaggggt cggcaattga tccggtgcct agagaaggtg gcgcggggta aactgggaaa 780

gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg 840

cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggttggac 900

cggtgccacc atggactata aggaccacga cggagactac aaggatcatg atattgatta 960

caaagacgat gacgataaga tggcccccaa aaagaaacga aaggtgggtg ggtccccaaa 1020

gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt acagcatcgg 1080

cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc 1140

cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat 1200

cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc 1260

cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa 1320

cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga 1380

agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta 1440

ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa 1500

ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt 1560

cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct 1620

ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc 1680

caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca 1740

gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct 1800

gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa 1860

ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga 1920

cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt 1980

gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca 2040

ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa 2100

agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag 2160

ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga 2220

actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg 2280

cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga 2340

tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat 2400

cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa 2460

gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc 2520

ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct 2580

gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa 2640

atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat 2700

cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta 2760

cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa 2820

cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga 2880

caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga 2940

cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat 3000

gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa 3060

cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt 3120

cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat 3180

ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc 3240

cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt 3300

gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca 3360

gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat 3420

caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa 3480

cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact 3540

ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa 3600

ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga 3660

caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa 3720

cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct 3780

gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac 3840

aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa 3900

gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa 3960

ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta 4020

cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt 4080

cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga 4140

aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac 4200

cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga 4260

aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag 4320

catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga 4380

gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc 4440

taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa 4500

agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat 4560

catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa 4620

agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa 4680

cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct 4740

gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc 4800

ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga 4860

gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga 4920

caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa 4980

tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga 5040

caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat 5100

ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga 5160

caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagg gcggctccaa 5220

gcggcctgcc gcgacgaaga aagcgggaca ggccaagaaa aagaaaggat ccggcgcaac 5280

aaacttctct ctgctgaaac aagccggaga tgtcgaagag aatcctggac cggtgagcaa 5340

gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa 5400

cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac 5460

cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac 5520

cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt 5580

cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga 5640

cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat 5700

cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta 5760

caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt 5820

gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca 5880

gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac 5940

ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt 6000

cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagggct ccggcgaggg 6060

caggggaagt cttctaacat gcggggacgt ggaggaaaat cccggcccaa ccgagtacaa 6120

gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc 6180

cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg 6240

ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg 6300

ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg 6360

ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc 6420

gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct 6480

ggccaccgtc ggagtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct 6540

ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc 6600

ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc 6660

cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgaacgc gttaagtcga 6720

caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc 6780

tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg 6840

tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt 6900

gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac 6960

tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc 7020

tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct 7080

gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct 7140

cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct 7200

caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct 7260

tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc gtcgacttta 7320

agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga 7380

ctggaagggc taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct 7440

ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 7500

aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 7560

tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagggcc 7620

cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg 7680

cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata 7740

aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt 7800

ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt 7860

gggctctatg gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt 7920

atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg 7980

cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg 8040

ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 8100

taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 8160

aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 8220

ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac 8280

tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt 8340

ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt 8400

ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc 8460

cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg 8520

cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat 8580

caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca 8640

tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc 8700

ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct 8760

gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg 8820

cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg 8880

tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc 8940

tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca 9000

cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac 9060

tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa 9120

agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg 9180

ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt 9240

ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg 9300

aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc 9360

gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga 9420

tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta 9480

ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc 9540

cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg 9600

atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt 9660

cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa 9720

ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt 9780

cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt 9840

ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt 9900

tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga 9960

taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag 10020

caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata 10080

agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg 10140

gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga 10200

gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca 10260

ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa 10320

acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt 10380

tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac 10440

ggttcctggc cttttgctgg ccttttgctc acatgt 10476

<210> 3

<211> 3120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60

cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 120

tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 180

aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 240

ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 300

ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 360

tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 420

tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 480

actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 540

gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 600

acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 660

gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 720

acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 780

gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 840

ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 900

gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 960

cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1020

agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1080

catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1140

tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1200

cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1260

gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1320

taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 1380

ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1440

tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1500

ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 1560

cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1620

agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1680

gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1740

atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1800

gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1860

gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1920

ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 1980

cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 2040

cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 2100

acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 2160

cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 2220

accatgatta cgccaagctt gcatgcaggc ctctgcagtc gacgggcccg ggatccgatg 2280

ataaacatgt gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc 2340

tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac 2400

gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat 2460

ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt 2520

gtggaaagga cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag 2580

ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttc 2640

tagcgcgtgc gccaattctg cagacaaatg gctctagagg tacccataga tctagatgca 2700

ttcgcgaggt accgagctcg aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa 2760

accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta 2820

atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat 2880

ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt 2940

gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa 3000

cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg 3060

tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga 3120

<210> 4

<211> 175

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

tgtggaaagg acgaaacacc gggtcttcga gaagacctgt tttagagcta gaaatagcaa 60

gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt 120

ctagcgcgtg cgccaattct gcagacaaat ggctctagag gtacccgtta cataa 175

<210> 5

<211> 554

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

tctgcagaca aatggctcta gaggtacccg ttacataact tacggtaaat ggcccgcctg 60

gctgaccgcc caacgacccc cgcccattga cgtcaatagt aacgccaata gggactttcc 120

attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 180

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 240

gtgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 300

tcgctattac catgggggca gagcgcacat cgcccacagt ccccgagaag ttggggggag 360

gggtcggcaa ttgatccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg 420

tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag 480

tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggtt ggaccggtgc 540

caccatggac tata 554

<210> 6

<211> 447

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

ccagaacaca ggttggaccg gtgccaccat ggactataag gaccacgacg gagactacaa 60

ggatcatgat attgattaca aagacgatga cgataagatg gcccccaaaa agaaacgaaa 120

ggtgggtggg tccccaaaga agaagcggaa ggtcggtatc cacggagtcc cagcagccga 180

caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg ccgtgatcac 240

cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg accggcacag 300

catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag ccgaggccac 360

ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga tctgctatct 420

gcaagagatc ttcagcaacg agatggc 447

<210> 7

<211> 2727

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa gggcggctcc aagcggcctg 60

ccgcgacgaa gaaagcggga caggccaaga aaaagaaagg atccggcgca acaaacttct 120

ctctgctgaa acaagccgga gatgtcgaag agaatcctgg accggtgagc aagggcgagg 180

agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca 240

agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt 300

tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct 360

acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt 420

ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact 480

acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga 540

agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca 600

acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca 660

agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca 720

cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg 780

ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg 840

ccgccgggat cactctcggc atggacgagc tgtacaaggg ctccggcgag ggcaggggaa 900

gtcttctaac atgcggggac gtggaggaaa atcccggccc aaccgagtac aagcccacgg 960

tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg 1020

ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg 1080

agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg 1140

acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt 1200

tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac 1260

agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg 1320

tcggagtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag 1380

tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc 1440

tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac 1500

cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaac gcgttaagtc gacaatcaac 1560

ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta 1620

cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt 1680

tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg 1740

ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg 1800

gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca 1860

cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca 1920

ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg 1980

ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag 2040

cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc 2100

gccctcagac gagtcggatc tccctttggg ccgcctcccc gcgtcgactt taagaccaat 2160

gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg 2220

gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt ctctctggtt 2280

agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca 2340

ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa 2400

ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcaggg cccgtttaaa 2460

cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc 2520

ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg 2580

aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg 2640

acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta 2700

tggcctgcag gggcgcctga tgcggta 2727

<210> 8

<211> 410

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

gataaacatg tgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg 60

ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata 120

cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa 180

tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct 240

tgtggaaagg acgaaacacc gggtcttcga gaagacctgt tttagagcta gaaatagcaa 300

gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt 360

ctagcgcgtg cgccaattct gcagacaaat ggctctagag gtacccatag 410

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

agttatggca gaactcagtg 20

<210> 10

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

ccccatccaa agtttttaaa gga 23

<210> 11

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

tgtggcagat gtcacagttt agg 23

<210> 12

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

caccgagtta tggcagaact cagtg 25

<210> 13

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

aaaccactga gttctgccat aactc 25

<210> 14

<211> 1180

<212> DNA

<213> pig (Sus scrofa)

<400> 14

ttttggcccc tgatagtttg catttcccct aactcagtat ttagtcctga gcccatctaa 60

ctgattaaac tagctcatct gatgattatt atcaaattag tatttccagt ttggacctta 120

ttccttaagc agatcatttt tcatttcctt tgaaaatctt attgtgaagt acatcactgt 180

ttcagaatca gttaccatgt agaacaatat gttttctctg catggaaagt aatataaatt 240

gcatcccacc ttacactagg aaacagattg ttgttaccag tacatccaca gctcatgcac 300

atgtacatcc tgaaaatgtg agttgaatat tgtcttcatt gttttattaa gaccaaatat 360

gaaaaatgca tgaagtaaat aatctttagt cttatgaaaa atgtcaaagg aatctaggag 420

aaatcttgca aattcttgag caaagcaata aaaactttta aacactatta acttggttga 480

tcccttgtgt cttcttatag atgaacatgg tcatgtccct cctggggatg ttctgtccaa 540

cactgtttga tttatttgct gaattggaag actatcatcc cctcatcgct ctgaaatggc 600

tactgggacg catttttgct cttcttttag gcaacttgta tgtatttatt cttgcattga 660

tggatgagat taacaacaag gtaaatcttg tgtctggatt gtgcttgaaa tcagcatatt 720

gttatattcc cagtctcaag cttgccagaa attacttgga aatctgtgtt gtcttcattt 780

ttgttttcat cactctgctc tctagattga agaagagaag ctagtaaagg ccaatattac 840

cttatgggaa gccaacatga tcaaggccta caatgcatcg ctcactggaa atagcactgg 900

gccacccttt tttgtacatc cggcagatgt acctcgagga ccctgctggg aaacgatggt 960

ggggcaggta atgtcaaaaa cagaagtccg ttaacaatta atgcattaaa aaagagcaga 1020

gttcatattt cttccatatt gtgacctgtg tttttaattg cttattaatg aaaatagcta 1080

agatttaccg acagcttgcc atatgctggt tatgaatttc atatgtatta tctcatgcag 1140

tcctcatatg catcctttca ggtaagtaga acatttacac 1180

<210> 15

<211> 759

<212> PRT

<213> pig (Sus scrofa)

<400> 15

Met Pro Pro Lys Lys Val Gln Ile Gln Val Glu Glu Lys Glu Asp Asp

1 5 10 15

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

20 25 30

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

35 40 45

Glu Asp Asp Pro Glu Pro Glu Pro Glu Asp Glu Glu Thr Arg Lys Ala

50 55 60

Arg Glu Lys Glu Arg Arg Arg Arg Leu Arg Arg Gly Ala Glu Glu Glu

65 70 75 80

Glu Glu Ile Asp Glu Glu Glu Leu Glu Arg Leu Lys Ala Glu Leu Asp

85 90 95

Glu Lys Arg Gln Met Ile Ala Thr Val Lys Cys Lys Pro Trp Lys Met

100 105 110

Glu Lys Lys Ile Glu Val Leu Lys Glu Ala Lys Lys Phe Val Ser Glu

115 120 125

Asn Glu Gly Ala Leu Gly Lys Gly Lys Gly Lys Gln Trp Phe Ala Phe

130 135 140

Lys Met Met Met Ala Lys Lys Trp Ala Lys Phe Leu Arg Asp Phe Glu

145 150 155 160

Asn Phe Lys Ala Ala Cys Ile Pro Trp Glu Asn Lys Ile Lys Ala Ile

165 170 175

Glu Ser Gln Phe Gly Ser Ser Val Ala Ser Tyr Phe Leu Phe Leu Arg

180 185 190

Trp Met Tyr Gly Val Asn Met Val Leu Phe Ile Leu Thr Phe Ser Leu

195 200 205

Ile Met Leu Pro Glu Tyr Leu Trp Gly Leu Pro Tyr Gly Ser Leu Pro

210 215 220

Arg Lys Thr Val Pro Arg Ala Glu Glu Ala Ser Ala Ala Asn Phe Gly

225 230 235 240

Val Leu Tyr Asp Phe Asn Gly Leu Ala Gln Tyr Ser Val Leu Phe Tyr

245 250 255

Gly Tyr Tyr Asp Asn Lys Arg Thr Ile Gly Trp Met Asn Phe Arg Leu

260 265 270

Pro Leu Ser Tyr Phe Leu Val Gly Ile Met Cys Ile Gly Tyr Ser Phe

275 280 285

Leu Val Val Leu Lys Ala Met Thr Lys Asn Ile Gly Asp Asp Gly Gly

290 295 300

Gly Asp Asp Asn Thr Phe Asn Phe Ser Trp Lys Val Phe Thr Ser Trp

305 310 315 320

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

325 330 335

Ile Thr Met Asn Phe Lys Glu Ala Ile Ile Glu Glu Arg Ala Ala Gln

340 345 350

Val Glu Glu Asn Val His Leu Ile Arg Phe Leu Arg Phe Leu Ala Asn

355 360 365

Phe Phe Val Phe Leu Thr Leu Gly Gly Ser Gly Tyr Leu Ile Phe Trp

370 375 380

Ala Val Lys Arg Ser Gln Glu Phe Ala Gln Gln Asp Pro Asp Thr Leu

385 390 395 400

Gly Trp Trp Glu Lys Asn Glu Met Asn Met Val Met Ser Leu Leu Gly

405 410 415

Met Phe Cys Pro Thr Leu Phe Asp Leu Phe Ala Glu Leu Glu Asp Tyr

420 425 430

His Pro Leu Ile Ala Leu Lys Trp Leu Leu Gly Arg Ile Phe Ala Leu

435 440 445

Leu Leu Gly Asn Leu Tyr Val Phe Ile Leu Ala Leu Met Asp Glu Ile

450 455 460

Asn Asn Lys Ile Glu Glu Glu Lys Leu Val Lys Ala Asn Ile Thr Leu

465 470 475 480

Trp Glu Ala Asn Met Ile Lys Ala Tyr Asn Ala Ser Leu Thr Gly Asn

485 490 495

Ser Thr Gly Pro Pro Phe Phe Val His Pro Ala Asp Val Pro Arg Gly

500 505 510

Pro Cys Trp Glu Thr Met Val Gly Gln Glu Phe Val Arg Leu Thr Val

515 520 525

Ser Asp Val Leu Thr Thr Tyr Val Thr Ile Leu Ile Gly Asp Phe Leu

530 535 540

Arg Ala Cys Phe Val Arg Phe Cys Asn Tyr Cys Trp Cys Trp Asp Leu

545 550 555 560

Glu Tyr Gly Tyr Pro Ser Tyr Thr Glu Phe Asp Ile Ser Gly Asn Val

565 570 575

Leu Ala Leu Ile Phe Asn Gln Gly Met Ile Trp Met Gly Ser Phe Phe

580 585 590

Ala Pro Ser Leu Pro Gly Ile Asn Ile Leu Arg Leu His Thr Ser Met

595 600 605

Tyr Phe Gln Cys Trp Ala Val Met Cys Cys Asn Val Pro Glu Ala Arg

610 615 620

Val Phe Lys Ala Ser Arg Ser Asn Asn Phe Tyr Leu Gly Met Leu Leu

625 630 635 640

Leu Ile Leu Phe Leu Ser Thr Met Pro Val Leu Tyr Met Ile Val Ser

645 650 655

Leu Pro Pro Ser Phe Asp Cys Gly Pro Phe Ser Gly Lys Asn Arg Met

660 665 670

Phe Glu Val Ile Gly Glu Thr Leu Glu His Asp Phe Pro Ser Trp Met

675 680 685

Ala Lys Ile Leu Arg Gln Leu Ser Asn Pro Gly Leu Val Ile Ala Val

690 695 700

Val Leu Val Met Ala Leu Thr Ile Tyr Tyr Leu Asn Ala Thr Ala Lys

705 710 715 720

Gly Gln Lys Ala Ala Asn Leu Asp Leu Lys Lys Lys Met Lys Gln Gln

725 730 735

Ala Leu Glu Asn Lys Leu Arg Asn Lys Lys Met Ala Ala Ala Arg Ala

740 745 750

Ala Ala Ala Ala Gly Ser Gln

755

<210> 16

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

gcatcccacc ttacactagg aa 22

<210> 17

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

cagagcgatg aggggatgat 20

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

ttggacagaa catccccagg 20

<210> 19

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

acatggtcat gtccctcctg 20

<210> 20

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

tggacagaac atccccagga 20

<210> 21

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

gtgttggaca gaacatcccc 20

<210> 22

<211> 100

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

uuggacagaa cauccccagg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 23

<211> 100

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 23

acauggucau gucccuccug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 24

<211> 100

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 24

uggacagaac auccccagga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 25

<211> 100

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 25

guguuggaca gaacaucccc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 26

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 26

caccgttgga cagaacatcc ccagg 25

<210> 27

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 27

aaaccctggg gatgttctgt ccaac 25

<210> 28

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 28

caccgacatg gtcatgtccc tcctg 25

<210> 29

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 29

aaaccaggag ggacatgacc atgtc 25

<210> 30

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 30

caccgtggac agaacatccc cagga 25

<210> 31

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 31

aaactcctgg ggatgttctg tccac 25

<210> 32

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 32

caccgtgttg gacagaacat cccc 24

<210> 33

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 33

aaacggggat gttctgtcca acac 24

Claims (8)

1. A gRNA for TMC1 gene editing, characterized by a sequence as set forth in SEQ ID NO: 25.

2. A gRNA expression vector for pig TMC1 gene, characterized in that the gRNA of claim 1 is expressed by taking pKG-U6gRNA with the complete sequence shown as SEQ ID NO.3 as a vector skeleton.

3. The expression vector according to claim 2, characterized in that SEQ ID NO:32 and SEQ ID NO:33, and a double-stranded DNA molecule having a cohesive end obtained by annealing the single-stranded DNA shown in FIG. 33 is inserted into the BbsI restriction enzyme site of the vector backbone pKG-U6 gRNA.

4. A CRISPR/Cas9 composition for pig TMC1 gene editing, characterized by comprising a Cas9 expression vector and the gRNA expression vector for pig TMC1 gene of claim 2 or 3.

5. The CRISPR/Cas9 composition according to claim 4, characterized in that the Cas9 expression vector is a pU6gRNA-eEF1 a-mNLS-hscas 9-EGFP-PURO vector with a plasmid full sequence as shown in SEQ ID No. 2.

6. The CRISPR/Cas9 composition according to claim 5, characterized in that the molar ratio of the gRNA expression vector to Cas9 expression vector is 1-3:1.

7. The CRISPR/Cas9 composition according to claim 6, characterized in that the molar ratio of gRNA expression vector to Cas9 expression vector is 3:1.

8. Use of the CRISPR/Cas9 composition of any one of claims 4 to 7 for constructing an congenital genetic deafness clone pig nuclear donor cell.