CN112094866B - Method for preparing CD163 gene editing pig by using SpRY-Cas9 system - Google Patents

Abstract

The invention discloses a method for preparing a CD163 gene editing pig by using a SpRY-Cas9 system. The invention provides a method for preparing CD163 biallelic gene mutant cells, which is to carry out gene editing on CD163 genes of pig in vitro fibroblast genomes, so that GTCGAGGGAATGA at the 226 th and 238 th sites of the third exon of one chromosome of the biallelic gene CD163 is deleted, GTCGAGGGAAT at the 226 th and 236 th sites of the third exon of the other chromosome is deleted, and other nucleotide sequences are not changed, thus obtaining the CD163 biallelic gene mutant cells; the CD163 double allele knockout pig prepared by the cell has the capability of resisting the PRRSV, which shows that the method for preparing the CD163 double allele knockout pig can obtain the PRRSV virus resisting pig.

Description

Method for preparing CD163 gene editing pig by using SpRY-Cas9 system

Technical Field

The invention belongs to the technical field of biology, and relates to a method for preparing a CD163 gene editing pig by using a SpRY-Cas9 system.

Background

Porcine Reproductive and Respiratory Syndrome (PRRS) is a global, highly contagious, and highly dangerous viral disease of swine with clinically varying symptoms in pigs of all ages, but primarily resulting in late abortions, stillbirths, and mummification of pregnant sows and respiratory disease in piglets, with high mortality, significant economic losses to the swine industry worldwide, e.g., PRRS incurs a total of $ 6.64 billion per year in the united states, a 15 billion euro loss in europe, and a loss of perhaps 70 billion RMB in china.

The disease first developed in the united states in 1987, and then rapidly spread to the world in 1990-1992. The causative agent is Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), which is mainly of two types I and II. PRRSV is a single-stranded positive sense RNA virus that is a member of the order of the nested virus (Nidovirales), the family of arterividae (arterividae), the genus of Arterivirus (Arterivirus), has a genome of 15kb in length, and contains 11 open reading frames in total. It was found that the only natural host for PRRSV is a pig, which can infect differentiated blood mononuclear cells and porcine alveolar macrophages in the pig. Calvert et al found CD163 by screening a cDNA library constructed from porcine alveolar macrophages for genes susceptible to PRRSV, and CD163 was expressed only in the monocyte/macrophage lineage. Scientists then expressed CD163 in several non-susceptible PRRSV cell lines, which became susceptible to PRRSV, and the ability of PPRSV to infect these cell lines was positively correlated with the amount of CD163 expression, indicating that it is an important receptor for mediating PRRSV infection.

With the recent development of genome editing technology (especially CRISPR/Cas 9) and the discovery of the important role of CD163 gene in PRRSV infection, it is possible to improve the breed of large animals by using the gene editing technology. The first world CD163 knockout pig was prepared by Randall S. Prather group of university of Missouri and Genus, UK, by the cooperation of CRISPR/Cas9 technology in combination with either prokaryotic injection or somatic cloning technology. Subsequently in 2016, they performed challenge experiments to demonstrate that CD163 knockout pigs are resistant to type II PRRSV, with related results published in Nature biotechnology journal. The topic group of Wuzhenhuang professor at national south China agricultural university in 2017 also successfully prepares the CD163 gene knockout pig resisting the domestic HF-PRRSV by using the CRISPR/Cas9 technology in combination with the cell cloning technology. The Randall s, Prather project group produced CD163 knockout pigs and E7 exon replacement pigs in 2018 with the british plus company. In the same year, CD163 gene knockout pigs were successfully prepared by using CRISPR/Cas9 technology in combination with cell cloning technology in professor Liquai, Beijing animal husbandry and veterinary institute of Chinese academy of agricultural sciences. However, although the CD163 gene knockout can resist different types of PRRSV, since the CD163 protein has other important biological functions such as clearing hemoglobin, erythrocyte adhesion receptor, immunoregulatory factor, etc. in plasma besides mediating PRRSV infection, the CD163 gene knockout may affect other functions of pigs, and if only the SRCR5 functional domain of the protein encoded by the CD163 gene is deleted, the PRRSV virus infection is prevented, and other functional domains of the protein encoded by the CD163 gene are retained, so that the gene editing pigs can resist PRRSV, and other physiological functions of pigs can not be affected. In 2017, Bruce A. Whitelaw subject group and British Genus company cooperate to successfully prepare the 7 th exon deleted pig of the first CD163 gene in the world, the positive pig cell level resists the PRRSV viruses I and II, and the important functions of normal hemoglobin clearance of CD163 and the like are kept. The pig with the CD163 gene E7 exon accurately replaced and deleted is successfully prepared by using a CRISPR/Cas 9-mediated homologous recombination technology and a somatic cell cloning technology by Huxiaxiang professor of China agricultural university in 2019. A pig with a deletion of the E7 exon part of the CD163 gene was prepared by using CRISPR/Cas9 technology in combination with cell cloning technology by the assistant professor of Ohio university in Zhongshan of the same year.

However, this technique has mainly 2 defects at present: firstly, the off-target phenomenon exists, and the off-target effect is greatly reduced by methods such as upgraded nickase version dCas9, high-fidelity Cas9, dCas9-forkI, gRNA modification and the like. Second, the limitations of PAM make it difficult for researchers to freely design and edit candidate gene regions. For example, it is advantageous to design a gene knock-out to hope to knock out a target gene, and at the same time, to make the target gene encode a small number of AA, so as to study the residual protein in vivo. For example, the introduction of knock-out near ATG results in only a few AA for the gene of interest, security certificate declaration for commercialization, and the like.

The Benjamin P. Kleinstimer subject group successfully prepared the SpRY-Cas9 technology without PAM limitation aiming at optimizing the technical defects in 2020 and successfully verified in HEK 293T cells.

Disclosure of Invention

It is an object of the present invention to provide a method for preparing a CD163 biallelic mutant cell.

The method provided by the invention is to carry out gene editing on the CD163 gene of the porcine in vitro fibroblast genome, so that GTCGAGGGAATGA at the 226-238 th site of the third exon of one chromosome of the biallelic gene CD163 is deleted, GTCGAGGGAAT at the 226-236 th site of the third exon of the other chromosome is deleted, and other nucleotide sequences are not changed, thus obtaining the CD163 biallelic gene mutant cell;

the nucleotide sequence of the third exon (E3 exon) is sequence 2.

In the method, the gene editing is carried out by adopting a SpRY-Cas9 system;

the SpRY-Cas9 system includes Cas9 protein and sgRNA;

the target sequence of the sgRNA is 1 st-20 th of the sequence 3;

the cas9 protein is a protein coded by nucleotides from 20 th to 4204 th in the sequence 6.

In the above method, the SpRY-Cas9 system is 1) or 2) as follows:

1) consisting of the following 1) -A and 1) -B):

1) -a is said cas9 protein or mRNA thereof or a recombinant vector expressing the protein;

1) -B is the sgRNA; the nucleotide sequence of the sgRNA is sequence 3 or sequence 8 in the sequence table;

2) a vector for expressing the cas9 protein and the sgRNA.

In the method, the gene editing is to introduce the cas9 protein mRNA and the sgRNA into the in vitro pig fibroblast cell line to obtain the CD163 double-allele mutant cell.

Another objective of the invention is to provide a method for preparing a CD163 double allele mutant pig.

According to the method provided by the invention, the CD163 double allele mutant cell prepared by the first objective method is transplanted into a sow body through a somatic cell nucleus, and the produced offspring is the CD163 double allele mutant pig.

The 3 rd object of the invention is to provide a method for preparing pigs resisting the porcine reproductive and respiratory syndrome virus.

The method provided by the invention is 1) or 2):

1) transplanting the CD163 double allele mutant cell prepared by the 1 st purpose into a sow through somatic cell nucleus to produce a filial generation, namely a CD163 double allele mutant pig, namely a pig resisting the porcine reproductive and respiratory syndrome virus;

2) and (3) carrying out propagation expansion on the CD163 double-allele mutation pig to obtain the pig resisting the porcine reproductive and respiratory syndrome virus.

It is still another object of the present invention to provide a CD163 biallelic mutant gene.

The CD163 biallelic mutant gene provided by the invention is obtained by deleting GTCGAGGGAATGA at position 226-238 of the third exon of one chromosome of the biallelic gene CD163, deleting GTCGAGGGAAT at position 226-236 of the third exon of the other chromosome and keeping other nucleotide sequences unchanged;

the nucleotide sequence of the E3 exon is sequence 2.

It is still another object of the present invention to provide a biological product for preparing pigs resistant to porcine reproductive and respiratory syndrome virus.

The biological product provided by the invention is a biological product with unchanged other nucleotide sequences, wherein GTCGAGGGAATGA at the 226-238 th site of the third exon of one chromosome of the biallelic gene CD163 in a pig genome is deleted, GTCGAGGGAAT at the 226-236 th site of the third exon of the other chromosome is deleted;

the nucleotide sequence of the third exon is sequence 2.

In embodiments of the invention, the biologic is the SpRY-Cas9 system described in the methods above.

The application of the method in preparing the porcine reproductive and respiratory syndrome virus resistant pigs is also within the protection scope of the invention;

the application of the biological product in preparing the porcine reproductive and respiratory syndrome virus resistant pigs is also within the protection scope of the invention;

or, the application of the biological product in breeding pigs resisting the porcine reproductive and respiratory syndrome virus is also within the protection scope of the invention.

The pig is especially white pig.

The SpRY-Cas9 system includes a Cas9 protein and an sgRNA that includes a binding region that binds a target sequence and a binding region that binds a Cas9 protein.

The mRNA of cas9 protein is RNA transcribed from the nucleotide sequence consisting of the 20 th to 4204 th positions in SEQ ID No. 6, and the protein is encoded by the nucleotides consisting of the 20 th to 4204 th positions in SEQ ID No. 6.

The invention has the following advantages:

1) mutation of E3 exon region of CD163 Gene

Mutations to the existing Cas9 technology are all CD163 gene E7 exons, but mutations to the E7 exon result in a CD163 gene that is too long to produce a truncated protein of unknown function, which is not known to have a potential risk for either functioning of the PRRSV virus or having an effect on the host in vivo. The inventor successfully carries out frame-shift deletion mutation on the E3 exon region of the CD163 gene by using the latest SpRY-Cas9 technology for the first time to cause AA termination, so that the protein translation is terminated in advance to achieve the purpose of gene knockout, and the CD163 gene editing pig is successfully prepared.

2) Transfection with mRNA

The possibility that an exogenous SpRY-Cas9 plasmid is integrated into a receptor genome exists when SpRY-Cas9 plasmid is used for preparing a gene editing animal, so that mRNA of SpRY-Cas9 is used for transfection, the problem is avoided, the biological safety problem caused by screening of marker genes is avoided, then a screening marker-free gene editing method is used, CD163 double-allele mutation positive cell clone without exogenous DNA integration is finally obtained, 13bp deletion and 11bp deletion mutation of CD163 gene third exons on two chromosomes cause frameshift mutation, AA is caused to terminate in advance, and the purpose of CD163 gene knockout is achieved. The positive cells are subjected to somatic cloning to prepare the CD163 gene knockout pig with accurate editing. Experiments demonstrated that CD163 protein is not present in the PAM cells of this pig.

The invention firstly establishes a method for successfully preparing a CD163 knock-out pig by using a novel Cas9 technology without PAM sequence restriction to cause frame shift mutation immediately after ATG of a gene, and specifically knocking out the gene in the region near an E3 exon ATG of the CD163 gene. This method not only results in gene knock-out, but also leaves little AA in the target gene. The method greatly improves the operability and the universality of target gene editing of designers, and simultaneously can ensure that the target gene codes small protein and then degrades, so that the residual protein of the target gene does not influence the functions of animals. The invention provides important technical support for improving the freedom and the universality of gene editing and gene knockout design of pigs.

The CD163 double allele knockout pig obtained by the invention has the capability of resisting PRRSV virus, which shows that the method for preparing the CD163 double allele knockout pig can obtain the PRRSV virus resisting pig, the obtained CD163 double allele knockout pig can be used as a disease resisting pig for propagation expansion, somatic cell nucleus transplantation propagation of the ear-like somatic cell of the CD163 double allele knockout pig can be also used, and the CD163 double allele pig fibroblast mutant can be also used for the somatic cell nucleus transplantation propagation of the disease resisting pig.

Drawings

FIG. 1 is a technical scheme of the present invention.

Figure 2 is a CD163 specific SpRY-Cas9 efficiency validation.

FIG. 3 is a TA clone sequencing alignment analysis of CD163 specific SpRY-Cas9-sgRNA 1.

FIG. 4 shows the sequencing results and phenotype of CD163 double allele knock-out pigs.

FIG. 5 shows the result of Western blot detection of CD163 double allele knockout porcine PAMs.

FIG. 6 shows the detection of the relative expression of viral RNA in PAMs under different challenge doses of PRRSV.

Figure 7 is the results of virus titer detection in PAMs at different challenge doses of PRRSV.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

pCMV-SpRY-2A-EGFP vector: addgen, Cat number: #139989, the vector comprises a Cas9 protein expression unit.

The SpRY-Cas9 system includes a Cas9 protein and an sgRNA that includes a binding region that binds a target sequence and a binding region that binds a Cas9 protein.

The mRNA of cas9 protein is RNA transcribed from the nucleotide sequence consisting of the 20 th to 4204 th positions in SEQ ID No. 6, and the protein is encoded by the nucleotides consisting of the 20 th to 4204 th positions in SEQ ID No. 6.

Example 1 preparation of CD163 Diallelic mutant cell line Using the SpRY-Cas9 System

This example utilizes the SpRY-Cas9 system to prepare a CD163 biallelic mutant cell line. Considering the later industrialization and avoiding the biosafety problem, the invention does not use a screening marker gene and simultaneously uses mRNA of Cas9 protein, thus preparing a CD163 biallelic gene mutant cell line without exogenous DNA integration, and the technical route is shown in figure 1.

Substances for CD163 biallelic mutation

1. Expression vector SpRY-sgRNA

In this study, the exon 3 region of the CD163 gene was selected as the targeting site, the sequence of the CD163 gene is shown in SEQ ID NO. 1, and the sequence of exon 3 (E3 exon) is shown in SEQ ID NO. 2.

3 gRNAs were designed whose recognition sequences (target sequences of sgRNAs) were: gRNA-1: ATGTTTCTTGTCGAGGGAAT, respectively; gRNA-2: GATGGATCATGTTTCTTGTC; gRNA-3: GATCATGTTTCTTGTCGAGG are provided.

Synthesizing single-stranded oligonucleotides according to the designed sequences respectively by the following method:

gRNA-F1: CACCGATGTTTCTTGTCGAGGGAAT

gRNA-R1: AAACATTCCCTCGACAAGAAACATC

gRNA-F2: CACCGGATGGATCATGTTTCTTGTC

gRNA-R2: AAACGACAAGAAACATGATCCATCC

gRNA-F3: CACCGGATCATGTTTCTTGTCGAGG

gRNA-R3: AAACCCTCGACAAGAAACATGATCC

annealing each of the gRNAs-F and the gRNA-R to obtain a double-stranded DNA fragment gRNA1 with a sticky end, a double-stranded DNA fragment gRNA2 with a sticky end and a double-stranded DNA fragment gRNA3 with a sticky end;

the pCMV-SpRY-2A-EGFP (purchased from Addgene, which contains a Cas9 protein expression unit) vector was purifiedBbsI, carrying out enzyme digestion to recover fragments, and respectively connecting the double-stranded DNA fragment gRNA1 with the cohesive end, the double-stranded DNA fragment gRNA2 with the cohesive end and the double-stranded DNA fragment gRNA3 with the cohesive end into the recovered fragments to obtain SpRY-sgRNA1, SpRY-sgRNA2 and SpRY-sgRNA 3.

The expression vector SpRY-sgRNA1 of SpRY-Cas9 is a recombinant plasmidInserting double-stranded DNA fragment gRNA1 with cohesive end into pCMV-SpRY-2A-EGFP vectorBbsI, obtaining plasmids between enzyme cutting sites; the gRNA1 is fused with the cas9 protein binding region on the vector to co-express sgRNA1 for targeting the target sequence and binding the cas9 protein for cleavage of the target region. The plasmid contains a sgRNA1 encoding gene (the sequence is shown in sequence 3, wherein, the 1 st to 20 th nucleotides are binding regions of a target region, and the 21 st to 100 th nucleotides are binding regions of cas9 protein), and the sgRNA1 is expressed.

SpRY-Cas9 expression vector SpRY-sgRNA2 is formed by inserting double-stranded DNA fragment gRNA2 with cohesive ends into pCMV-SpRY-2A-EGFP vectorBbsI, obtaining plasmids between enzyme cutting sites; the gRNA2 co-expresses sgRNA2 with the cas9 protein binding region on the vector for targeting to a target sequence and binding to cas9 protein for cleavage of the target region. The plasmid contains a sgRNA2 encoding gene (the sequence is shown in sequence 4, wherein, the 1 st to 20 th nucleotides are binding regions of a target region, and the 21 st to 100 th nucleotides are binding regions of cas9 protein), and the sgRNA2 is expressed.

SpRY-Cas9 expression vector SpRY-sgRNA3 is formed by inserting double-stranded DNA fragment gRNA3 with cohesive ends into pCMV-SpRY-2A-EGFP vectorBbsI, obtaining plasmids between enzyme cutting sites; the gRNA3 co-expresses sgRNA3 with the cas9 protein binding region on the vector for targeting to a target sequence and binding to cas9 protein for cleavage of the target region. The plasmid contains a sgRNA3 encoding gene (the sequence is shown in sequence 5, wherein, the 1 st to 20 th nucleotides are a target region binding region, and the 21 st to 100 th nucleotides are a region binding with cas9 protein), and the sgRNA3 is expressed.

The SpRY-Cas9 system comprises Cas9 protein and sgRNA, in the embodiment, the SpRY-Cas9 system is a vector for expressing Cas9 protein and sgRNA, and specifically can be a SpRY-Cas9 expression vector SpRY-sgRNA1, a SpRY-Cas9 expression vector SpRY-sgRNA2 or a SpRY-Cas9 expression vector SpRY-sgRNA 3.

2. Cutting efficiency verification

Cas9 mediates gene knock-out in mammalian cells, mainly by base deletion or base insertion of a small fragment formed by DNA double strand break, and this small fragment genetic modification can be detected by T7E1 cleavage. The basic principle is as follows: when the acting target is subjected to double-strand break, the cell can start a DNA repair mechanism to repair the acting target, so that various types of base deletion or insertion mutant types are generated. After a target sequence is amplified by PCR, due to deletion or insertion of partial bases, DNA bubbles can be generated by gradient annealing of different molecules through PCR, T7E1 can specifically identify the bubble positions and cut, and therefore whether cas9 can mediate gene knockout at a target site or not can be identified.

1) Designing PCR amplification primers aiming at recognition and editing position positions of SpRY-Cas9

PCR amplification primers F: 5 'AACATTTCTCAAATCTGG 3' and R: 5 'TTTCATGTAGAAGTAGAAGGT 3' were designed.

2) Preparation of pig fibroblast cell line

Removing hair from ear skin tissue of large white pig, cleaning with 70% (v/v) ethanol water solution, and picking with blade with area of 1cm²The skin was transported to the laboratory as soon as possible in DMEM/F12 medium at 0 deg.C, washed several times with PBS buffer and 70% (v/v) alcohol in water and chopped to 1mm³The left and right small blocks were washed with DMEM medium for 2 times and then planted in batches in 1mL DMEM medium flasks (25 cm in specification) containing 10% (v/v) FBS²) After the tissue blocks adhere firmly, the DMEM medium containing 10% (v/v) FBS is supplemented to 6mL, the temperature is 37 ℃, and the CO content is 5 percent₂Culturing in an incubator for 6-7 days, changing the culture solution every 2 days for 1 time, digesting and passaging for 2-3 times by 0.25% trypsin after the cells grow and converge, and freezing and storing by using a DMEM culture medium containing 20% (v/v) FBS and 10% (v/v) DMSO in batches to obtain the in vitro pig fibroblast line.

3) SpRY-Cas9 electrically transfected pig fibroblasts

(1) 2d before electrotransfer, 2X 10⁵The porcine fibroblast cell line obtained in 2) above was recovered in a 6-well plate culture dish and 4ml of DMEM medium (purchased from Gibco) containing 10% (volume percent) Fetal Bovine Serum (FBS) was added. Placing at 37 ℃ and 5% CO₂Culturing in an incubator.

(2) After the 6-well plate cells were full, about 1X 10⁶Cells were lysed with 1ml of 0.25% trypsin (from Gibco)Cells were liquid digested. The cells were pelleted by centrifugation at 1000g for 5 min. The cell pellet was washed 1 time with PBS buffer (purchased from Gibco). The cells were resuspended in 100. mu.L of shock buffer (purchased from LOZA, Inc.) to obtain a cell suspension.

(3) Mu.g of the expression vectors SpRY-sgRNA1, SpRY-sgRNA2 and SpRY-sgRNA3 prepared in the above 1 were added to 100. mu.L of the cell suspension obtained in the above (2), respectively, and the mixture was transferred to an electric shock cup (purchased from LOZA).

(4) Cells were shocked with an electric field strength of 1.2KV/cm and a pulse time of 1 ms.

(5) Transferring the cells after electric shock into a cell culture dish with the thickness of 60 mm; adding 4ml DMEM culture solution containing 10% (volume percentage) fetal calf serum into CO₂Culturing in an incubator, recovering the growth state of the cells, and respectively obtaining SpRY-sgRNA 1-transferred pig fibroblasts, SpRY-sgRNA 2-transferred pig fibroblasts and SpRY-sgRNA 3-transferred pig fibroblasts.

4) PCR amplification

Separately extracting genome DNA of the SpRY-sgRNA 1-transferred pig fibroblast 48h after the electrotransformation obtained in the step 3), SpRY-sgRNA 2-transferred pig fibroblast, SpRY-sgRNA 3-transferred pig fibroblast and the isolated pig fibroblast line (WT) prepared in the step 2).

Using genome DNA as a template, and using F and R designed and synthesized in the step 1) as primers to perform PCR amplification to obtain a PCR product.

5) Gradient annealing of PCR products

And (3) performing column purification and recovery on the PCR product obtained in the step 3), determining the concentration of the PCR product, and performing gradient annealing on the PCR product to obtain a gradient annealed PCR product.

The system of the gradient annealing is shown in the following table 1:

TABLE 1 gradient annealing system

System of	20 μL
		PCR product
	400 ng
		Taq Buffer (purchased from health as century)	2.0 μL
ddH2O	Make up to 20. mu.L system

The gradient annealing procedure is shown in table 2:

table 2 shows the PCR product gradient annealing procedure

Temperature of	Time	Ramp
			95°C	10min	-2°C
95°C-85°C	1 min	-0.3°C
			85°C-75°C	1 min	-0.3°C
75°C-65°C	1 min	-0.3°C
			65°C-55°C	1 min	-0.3°C
55°C-45°C	1 min	-0.3°C
			45°C-35°C	1 min	-0.3°C
35°C-25°C	1 min	-0.3°C
			4°C	Hold	—

6) The gradient annealed PCR product obtained in 5) above was subjected to T7E1 digestion, as shown in table 3 below:

table 3 shows the digestion system

System of	25 μL
		Gradient annealed PCR products	20 μL
Buffer 2.1(NEB)	2.5 μL
		T7E1(NEB)	0.5 μL
ddH2O	Make up to 25 μ L system

Reaction procedure: carrying out the enzyme digestion reaction shown in the table 3 in a constant temperature incubator at 37 ℃ for 1 h to obtain an enzyme digestion product.

Carrying out polyacrylamide gel (PAGE) electrophoresis detection on the enzyme digestion product, adopting 8% polyacrylamide gel, and after electrophoresis is finished, dyeing the PAGE gel by EB; and (3) carrying out gray level analysis on the enzyme digestion band through ImageJ software to preliminarily determine the SpRY-Cas9 cleavage efficiency.

The cutting efficiency is the strength ratio of the PCR main strip and the cut strip.

The upper panel of FIG. 2 shows the editing position of the gene.

The result of the cleavage efficiency test is shown in the lower graph of fig. 2, the percentage indicates the cleavage efficiency, WT is a pig fibroblast cell line (WT), SpRY-sgRNA1, SpRY-sgRNA2, SpRY-sgRNA3 correspond to a SpRY-sgRNA 1-transformed pig fibroblast, a SpRY-sgRNA 2-transformed pig fibroblast and a SpRY-sgRNA 3-transformed pig fibroblast, respectively; the results show that in the fibroblast of pig, the three grnas have recognition and cleavage activities aiming at the SpRY-Cas9 system corresponding to the three grnas 1, 2 and 3 of the CD163 locus, and the cleavage efficiencies are 20.1%, 13% and 15% respectively through analysis of biological gray scale analysis software.

In order to further determine that the mutation edited by the SpRY-Cas9 system is in the target region, SpRY-sgRNA1 transgenic pig fibroblasts with higher activity are mutextracted, genome DNA is used as a template, F and R primers are used for amplification, and the obtained PCR product is recovered and subjected to T-A clone sequencing.

The sequencing result is compared with the exon 3 (sequence 2) sequence of the wild CD163 gene, and the expected mutation of the sgRNA1 at the target site is confirmed (figure 3), so that the sgRNA1 can be an advantageous tool for mediating the mutation of the target gene, and the target sequence corresponding to the sgRNA1 is selected as a recognition region in subsequent experiments.

Second, preparation of CD163 double-allele mutant cell line by using SpRY-Cas9

Preparation of Cas9 protein mRNA in vitro transcription SpRY-Cas9

1. Room temperature configured in vitro transcription mRNA system 4

The pCMV-SpRY-2A-EGFP vector is used as a template, and T7-Cas9-F: ttaatacgactcactatagGGAGAATGGACTATAAGGACCACGAC and T7-Cas9-R: GCGAGCTCTAGGAATTCTTAC are used as primers for amplification to obtain a PCR product, namely a Cas9 encoding gene containing a T7 promoter (sequence 6, wherein the 20 th to 4204 th sequence 6 is a Cas9 encoding gene).

In vitro transcribed mRNA systems were prepared as shown in table 4; and then, after completely mixing the in vitro transcription mRNA system, incubating for 1 h at 37 ℃, adding 1 mu L of TURBO DNase into the reaction system, and incubating for 15min at 37 ℃ to obtain a reaction product.

TABLE 4 in vitro transcribed mRNA systems

Components	Volume of
		PCR product	1μL（0.1-1μg）
2×NTP/CAP	10μL
		10×Buffer	2μL
RNA synthetase	2μL
		ddH2O	Make up to 20 mu L

All of the above reagents were obtained from MEGAscript T7 Transcription Kit, Ambion, cat # SEQ ID NO: AM 1333.

2. Taking the reaction product obtained in the step 1, configuring an in vitro transcription mRNA and polyA system (table 5), mixing completely, and incubating at 37 ℃ for 1 h to obtain the reaction product.

Table 5 shows the addition of polyA

Components	Volume of
		Reaction product of step 1	20μL
5×E-PAP Buffer	20μL
		MnCl2(25mM)	10μL
ATP(10mM)	10μL
		E-PAP	4μL
ddH2O	36μL
		Total of	100μL

All from Poly (A) labeling Kit, Ambion, cat # K: AM 1350.

3. In vitro transcription of mRNA purification by column chromatography

(1) And (3) adding 350 mu L of binding buffer into the reaction product obtained in the step (2), blowing, sucking and uniformly mixing, transferring the sample into a column, and centrifuging at 10000g for 1min at room temperature.

(2) Discarding the filtrate, re-packing the column, rinsing the column with 500 μ L of eluent, centrifuging at 10000g at room temperature for 1min, and repeatedly rinsing once; the filtrate was discarded and centrifuged on an empty column for 15 s.

(3) The column was placed in a new centrifuge tube, 50. mu.L of the eluate was added to the center of the column, a lid was covered and incubated at 60 ℃ for 10min, 10000g was centrifuged at room temperature for 1min, and the supernatant was collected to obtain a Cas9 mRNA solution (concentration: 2. mu.g/. mu.L, in which the nucleotide sequence of Cas9 mRNA was the sequence obtained by substituting t of SEQ ID NO: 6 from 20 to 4204 with U).

Both the reagents and the column were from clear ­ Transcription clear-Up Kit, Ambion, cat #: AM 1908.

(II) preparation of in vitro transcription sgRNA

1. Artificially synthesized sgRNA1 in vitro transcription template

The SpRY-sgRNA1 vector is used as a template, T7-sgRNA-F: ttaatacgactcactatagggcaggactactcacactccg and T7-sgRNA-R: AAAAGCACCGACTCGGTGCC are used as primers for amplification, and a PCR product is obtained, namely a sequence containing a sgRNA1 encoding gene, wherein the nucleotide sequence is sequence 7, the 1 st to 20 th positions from the 5' end are a T7 promoter, the 19 th position is a transcription initiation site, the 21 st to 40 th positions are target sequences, and the 41 th to 120 th positions are binding regions of Cas9 protein in SpRY-Cas 9.

2. Taking the sequence containing the sgRNA1 encoding gene obtained in step 1, configuring an in vitro transcription system (table 6), and incubating at 37 ℃ for 2 h.

TABLE 6 in vitro transcription System

Components	Volume of
		Template shown in sequence 7	8 μL(2μg)
2×ATP/CTP/UTP/GTP	2/2/2/2μL
		10×Reaction Buffer	2 μL
Enzyme Mix
		2 μL
Total of			20 μL

The above reagents were all from MEGASHORTScript T7 Transcription Kit, AM1354, Ambion.

3. After completion of step 2, 1. mu.L of TURBO DNase (AM 1354, Ambion) was added to the reaction and incubated at 37 ℃ for 15 min.

4. After the step 3 is completed, 2 times of volume of absolute ethyl alcohol is added to precipitate the transcription product, the transcription product is kept stand at the temperature of minus 20 ℃ for 30min, the transcription product is centrifuged at 13000 rpm for 30min, the supernatant is discarded, and the precipitate is taken.

5. And (3) fully washing the precipitate obtained in the step (4) by using 75% (v/v) ethanol water solution, removing supernatant, fully drying by using an ultra clean bench, fully dissolving the sgRNA1 by using clean-free water to obtain a sgRNA1 solution (the concentration is 5 mu g/mu L, the sequence of the sgRNA1 is a sequence 8, and the 20 th-120 th nucleotide of the sequence 7 is transcribed to obtain a sequence).

All of the above reagents were obtained from MEGASHORTCRIPT-T7 Transcription Kit, Ambion, cat #: AM 1354.

(III), SpRY-Cas9 mRNA transfection

(1) 2 days before transfection, the isolated porcine fibroblast cell line obtained in step one, 2, was trypsinized into single cells and 1X 10 cells were added⁶Individual pig fibroblasts were transferred to a culture flask (100 mL size) and 4mL of DMEM medium (both from Gibco) containing 10% (v/v) fetal pig serum was added at 37 deg.C and 5% CO₂Culturing to logarithmic growth phase.

(2) After completion of step (1), porcine fibroblasts in logarithmic growth phase were taken, digested with 1ml of 0.25% trypsin (purchased from Gibco), and then centrifuged at 1000g for 5min, and the pellet was collected.

(3) After completion of step (2), the pellet was taken, washed 1 time with PBS buffer (from Gibco) and then resuspended with 100. mu.L of shock solution (from Loza, VPI 1002) to give a cell suspension.

(4) Taking cell suspension (containing 1X 10)⁶Individual cells), the SpRY-Cas9 mRNA solution obtained in the step (one) (making 4 μ g of SpRY-Cas9 mRNA in the transfection system) and the sgRNA1 solution obtained in the step (two) (making 4 μ g of sgRNA1 in the transfection system) were thoroughly mixed in the cell suspension, and then transferred into an electric shock cup for electric shock (electric field strength 1.2KV/cm, pulse time 1 ms).

(5) After the step (4) is completed, the cells after electric shock are transferred into a culture bottle (the specification is 100 mL), 4mL of DMEM culture solution containing 10% (v/v) fetal pig serum is added, the temperature is 37 ℃, and 5% CO is added₂After 2 days of culture, transfected cells were obtained.

(IV) preparation of single cell clone by infinite dilution method and genotype identification

(1) And (3) culturing the transfected cells obtained in the step (three) until the fusion rate reaches 80-90%, digesting the transfected cells by using 0.1% pancreatin at 37 ℃, stopping digestion by using a DMEM medium containing 10% (v/v) FBS, and centrifuging to collect the cells.

(2) The cells were resuspended in a DMEM medium containing 20% (v/v) FBS, a portion of the cells were counted, and the cells were diluted to 100 cells/mL to obtain a cell suspension. To 20 dishes to which 9mL of 20% (v/v) FBS-containing DMEM medium had been added, 1mL of each cell suspension was added at 37 ℃ with 5% CO₂And culturing under saturated humidity condition.

(3) When the cell clone in the culture dish grows to be more than 2mm in diameter, removing the culture medium, washing with DPBS, covering the clone cluster with a clone ring, dripping about 100 mu L of 0.1% pancreatin at 37 ℃ into the clone cluster, digesting for about 3min, dripping DMEM culture medium containing 20% (v/v) FBS to stop digestion, slightly blowing and then transferring the cell clone into a 48-well plate for culture expansion.

(4) When the cell fusion rate in the 48-well plate reaches 90%, half of the cells are taken for cell clone genotype identification after digestion, and the rest half are continuously cultured in the well plate.

(5) Centrifuging the cells for genotype identification at 1000g for 5min, discarding the supernatant, and adding 10-20 μ L cell lysate (50 Mm KCL, 2.5mM Mgcl) according to the cell precipitation₂10mM Tris-HCl, 0.45% NP40, 0.45% Tween 20 and 0.2mg/mL proteinase K).

(6) And 3 mu.L of cell lysate is taken as a template for PCR identification, and a primer pair consisting of a primer P1 and a primer P2 (which is designed and synthesized according to the porcine CD163 gene) is used for PCR amplification.

Primer P1: 5'-AACATTTCTCAAATCTGG-3', respectively;

primer P2: 5'-TTTCATGTAGAAGTAGAAGGT-3' are provided.

The reaction system was 20. mu.L, consisting of 1.0. mu.L of DNA template, 0.4. mu.L of primer P1 (10. mu.M), 0.4. mu.L of primer P2 (10. mu.M), 0.4. mu.L of dNTP, 0.3. mu.L of LA DNA polymerase, 2.0. mu.L of 10 XPCR Buffer, and 15.5. mu.L of ddH 2O.

Reaction procedure: 5min at 94 ℃; 30s at 94 ℃, 30s at 52 ℃,1min at 72 ℃ and 35 cycles; preserving at 72 deg.C for 5min and 4 deg.C.

A714 bp band was obtained.

And purifying and sequencing the PCR amplification product. And connecting the PCR product to a pMD-19t plasmid vector, transforming the competence of escherichia coli, selecting a plurality of single colonies (single cell clones) for sequencing, comparing the sequencing result with a3 rd exon (E3, sequence 2) of a wild type CD163 gene to obtain detailed mutation information, and determining whether the cell line is mutated or not and the mutation type.

The results are as follows:

of 39 single cell clones, 15 were single allele mutated, 3 were biallelic mutated: one cloning point is deleted at-6 bp/-19bp, one cloning point is deleted at-3 bp/-9 bp, and in the two cloning points, the mutation deletion type is the multiple of 3, so the frame shift mutation can not be formed, and the protein function deletion is caused; only one biallelic gene is deleted and premature amino acid termination due to the formation of frameshift mutations eventually leads to loss of protein function. The type of the biallelic gene mutation is biallelic gene-11/-13 bp deletion, the gene in the mutation form is named as CD163 biallelic mutant gene, and the cell containing the mutant gene is named as CD163 biallelic pig fibroblast mutant.

The CD163 biallelic mutant gene is the GTCGAGGGAATGA deletion at the position 226 and 238 of the third exon (sequence 2) of one chromosome of the biallelic gene of CD163, the GTCGAGGGAAT deletion at the position 226 and 236 of the third exon (sequence 2) of the other chromosome, and other nucleotide sequences are not changed.

Since the two strands of the allele of the CD163 biallelic mutant gene are deleted at the third exon-11/-13 bp, respectively, compared with the wild-type CD163 gene, the first exon before and the exon sequence after the deletion region are not changed, but due to deletion, the terminator appears in advance, so that the CD163 is terminated in advance, and the protein function is lost.

Example 2 preparation of CD163 Dual allele knockout pigs

Preparation of CD163 double allele knockout pig

1. The CD163 biallelic pig fibroblast mutant obtained in example 1 in the logarithmic growth phase was taken and digested with 0.25% trypsin for 5min to obtain single cells, which became donor cells in the subsequent operations.

2. Collecting ovaries of adult white pigs from slaughter houses, cleaning for three times in PBS (phosphate buffer solution) at 37 ℃, then extracting follicles with the diameter of 2-8mm by using a needle with the diameter of 0.7mm, recovering cumulus-oocyte-complex (COCs) with uniform shape and compact structure, washing for two times by using maturation liquid (M199+10% FBS +0.01U/mL bFSH +0.01U/mL bLH +1 mu g/mL estradiol), then putting the cumulus-oocyte-complex into a four-well plate containing the maturation liquid at 50-60/well, and cleaning for three times at 38.5 ℃ with 5% CO₂After the mature culture in the incubator for 18-20h, placing the mature oocyte into a tube of 0.1% hyaluronidase, oscillating for 2-3min, and lightly blowing and beating with a glass tube to completely separate the cumulus cell from the oocyte, and selecting the oocyte with complete shape, uniform cytoplasm and discharged first polar body as a cytoplasmic receptor (enucleated oocyte).

Transferring the oocyte with the first polar body into an operation liquid consisting of M199+10% (v/v) FBS +7.5 mug/mL cytochalasin B, cutting a small opening on the upper part of the polar body by using a glass needle under a 200-fold microscope, sucking chromosomes in the first polar body and the oocyte under the first polar body by using a glass tube with the inner diameter of 20 mug, washing the first polar body and the oocyte for three times by using M199 liquid containing 20% (v/v) FBS, and then placing the first polar body and the oocyte in an incubator for later use.

3. Transferring the donor cell obtained in the step 1 into the transparent band of the enucleated oocyte obtained in the step 2, firstly placing the donor cell into Zimmerman liquid (100 mL Zimmerman liquid is composed of 0.9854g of cane sugar, 10.7mg of magnesium acetate tetrahydrate, 1.8mg of calcium acetate monohydrate, 7.4mg of dipotassium hydrogen phosphate, 3.1mg of reduced glutathione, 1.0mg of porcine serum albumin and water) for balancing for 3min, then placing the donor cell into a fusion tank to rotate the oocyte to enable the donor cell to be in contact with the enucleated oocyte prepared in the step 2 and to be vertical to an electric field, and simultaneously placing the donor cell in a direct current pulse field with the field intensity of 2.5kV/cm, under the conditions of pulse duration of 10. mu.s, pulse frequency of 2 times, and pulse interval of 1s (the fusion apparatus is a product of BTX, model number of ECM-2001), the cells were rapidly transferred to M199 solution (a product of Gibco) containing 10% (v/v) FBS, and cultured at 37 ℃ with 5% CO2 for 30min to obtain reconstituted embryos.

3. Taking the reconstructed embryo obtained in the step 2, adding CR1aa culture solution (100 mL of CR1aa culture solution is composed of 0.67g of sodium chloride, 0.023g of potassium chloride, 0.22g of sodium bicarbonate, 2mg of sodium pyruvate, 100 mul of phenol red and water) of calcium ionophore A23178 with the concentration of 5 mul, and treating for 5 min; discarding the liquid phase, adding CR1aa culture solution containing 5 μ g/mL cytochalasin B and 10 μ g/mL cycloheximide, and treating for 5h (for activating recombinant embryo); discarding the liquid phase, adding CR1aa culture solution containing 5% (v/v) FBS, culturing at 37 deg.C and 5% CO2 for 48h, observing cleavage rate, and observing blastocyst development rate (about 80%) in 7-8 days to obtain transgenic cloned blastocysts.

4. The morphologically superior transgenic cloned blastocysts cultured for 7 days in step 3 were transferred into the uterine horn of a contemporary recipient sow (3 co-transplanted recipient sows). The recipient sows were subjected to B-ultrasonic examination at 30d after transplantation to determine the conception, and the pregnancy rate was 66.7%.

5. The pregnant sows are fed according to a conventional feeding method, and after 90 days, the pregnant sows give normal delivery to obtain somatic cell cloned pigs (namely CD163 double allele knockout pigs, wherein the number of normal survival piglets is #190910 pigs).

Detection of molecular level of II, CD163 double allele knockout pig

The genomic DNA of the ear tissues of the pigs to be tested (# 190910 pigs and wild white pigs) is extracted by adopting a blood and tissue genomic kit (QIAGEN company), and is used as a template, and a primer pair consisting of a primer P1 and a primer P2 (a primer P1: 5'-AACATTTCTCAAATCTGG-3'; a primer P2: 5'-TTTCATGTAGAAGTAGAAGGT-3') is adopted for PCR amplification, so that a PCR amplification product is obtained. And purifying and sequencing the PCR amplification product.

Sequencing results show that the genotype of the wild white pig (namely, the non-transgenic pig) based on the CD163 gene is wild type (CD 163 +/+), and the genotype of the #190910 pig based on the CD163 gene is CD163 biallelic mutant genotype (CD 163-/-).

The CD163 biallelic mutant genotype is characterized in that GTCGAGGGAATGA at the position 226 and 238 of the third exon (sequence 2) of one chromosome of the biallelic gene (sequence 1) of the wild type CD163 is deleted, GTCGAGGGAAT at the position 226 and 236 of the third exon (sequence 2) of the other chromosome is deleted, and other nucleotide sequences are not changed. This type of mutation causes premature termination of transcription and translation of mRNA of the CD163 gene, and thus loss of function of the CD163 protein.

The sequencing alignment results are shown in FIG. 4.

#190910 pig survived normally and developed normally to age 2, as shown in the phenotypic plot of FIG. 4.

Analysis of CD163 protein expression in PAMs (platelet-derived markers) cells of three-CD 163 double-allele knockout pigs

1. Establishment of PAMs

The CD163 double allele knockout pig (# 190910) obtained in the previous step was selected as a material source for establishing a CD163Mut/Mut Alveolar Macrophage System (PAMs), and wild type white pigs of the same day age, sex and breed were selected as a material source for establishing a WT alveolar macrophage system.

The preparation process of primary PAMs was as follows:

(1) anaesthetizing the pig, bleeding the carotid artery to die, opening the chest cavity by an operation, clamping the trachea by a hemostatic forceps to isolate the interior of the lung from the outside air, cutting off the trachea from the near-head end, taking out the whole lung, wrapping the lung by sterilized tin foil paper, and paying attention to no contamination of bacteria;

(2) transferring the lung to a clean bench, injecting HBSS buffer solution (Gibco) into the lung via trachea, kneading the lung, pouring out the injected liquid, and irrigating for 5-8 times;

(3) subpackaging the collected lavage liquid into 50 mL centrifuge tubes, centrifuging for 10min at 300 g, and discarding the supernatant;

(4) washing the collected cell sediment once by using HBSS buffer solution, then washing once by using RPMI-1640 culture solution (Gibco), and centrifuging again to collect the cell sediment;

(5) resuspending the cells with PAMs (platelet activating proteins) freezing medium (containing 90% FBS by volume percentage and 10% DMSO by volume percentage), freezing the cells in a gradient cooling mode, and finally storing in liquid nitrogen;

(6) when PAMs are resuscitated, the procedures for cell resuscitation are basically carried out according to the procedures for cell resuscitation, and the DMEM growth culture medium is only required to be replaced by RPMI1640 growth culture medium to obtain CD163Mut/Mut PAMs (CD 163 PAMs) and WT PAMs.

2. CD163 protein expression in PAMs cells

1) And extracting total protein from the CD163Mut/Mut PAMs (CD 163 PAMs) and WT PAMs obtained in 1 above.

2) And respectively taking the total protein obtained in the step 1) and carrying out Western blot. CD163 protein was detected using CD163 Antibody (product of AbD Serotec) as a primary Antibody and goat Antibody (product of China fir Jinqiao) as a secondary Antibody.

The results are shown in FIG. 5, and the total protein of the CD163 double allele knockout pig has no expression of CD163 protein, and the PAMs cells produced by the CD163 double allele knockout pig have no CD163 protein.

The results show that the porcine PAMs produced by the disruption of the CD163 gene in the CD163 double-allele knockout pig do not contain the CD163 protein. The swine individual is suggested to have the characteristic of resisting the porcine reproductive and respiratory syndrome virus.

anti-PRRSV infection analysis of CD163 double allele knockout pigs

1. Establishment of PAMs

The same procedure as for 1 of three gave CD163Mut/Mut PAMs (CD 163 PAMs) and WT PAMs.

2. Counteracting toxic substances

The PAMs obtained in 1 above were cultured in RPMI1640 medium containing 10% FBS and 4 Xdouble antibody by volume. After the cells are attached to the wall, the shape of the cells can be observed to be circular, and pseudopodia can be observed in part of the cells;

foreign PRRSV highly pathogenic strains typeII, NVSL97-7895 (Gene-edited pigs area protected from bacterial production and respiratory syndrome virus) were selected for in vitro infection CD163Mut/Mut PAMs and WT PAMs challenge tests, the former being referred to as experimental group and the latter as control group. All conditions were the same for both groups except that the source of PAMs used was different. Four different challenge doses were selected for each of the two groups of cells, with MOI = 0.005, MOI = 0.025, MOI = 0.1, and MOI = 0.25. Each group of cells was subjected to three replicates at each challenge dose, and a negative control was also run without added virus. And simultaneously thawing the cells of the experimental group and the cells of the control group, and simultaneously performing a toxicity counteracting test after culturing for 24 hours. Supernatant virus fluid and cells were collected 36 h post infection.

3. Detection of

The cells obtained in 2 above were lysed by TRIzol (Invitrogen) and used for total RNA extraction (QIAGEN RNeasy Mini Kit). The relative expression of viral RNA in Q-PCR cells was used.

cDNA obtained by reverse transcription (Thermo) of the obtained RNA is used as a template, and the relative expression quantity of the virus RNA in the collected cells is detected by utilizing fluorescent quantitative PCR. ORF7 of PRRSV is used as a detection target sequence, an upstream primer is ORF7-F (5 'AATAACAACGGCAAGCAGCA 3'), a downstream primer is ORF7-R (5 'GCACAGTATGATGCGTCGGC 3'), and a pig GAPDH-F (5 'GTCGGTTGTGGATCTGACCT 3') and a GAPDH-R (5 'GTCCTCAGTGTAGCCCAGGA 3') are used as internal reference primers. The Q-PCR reaction system is shown in Table 7 below:

table 7 shows the Q-PCR reaction system

System of	20 μL
		cDNA	1.0 μL
Forward primer (10 μM)	1.0 μL
		Reverse primer (10 μM)	1.0 μL
2 × SYBR green master mix (from Roche)	10.0 μL
		DEPC water	7.0 μL

The reaction procedure was as follows: 95 ℃ for 10min, 95 ℃ for 15s,60 ℃ for 1min, 40cycles, 95 ℃ for 15s,60 ℃ for 15s;

the results are shown in fig. 6, the relative expression quantity of the viral RNA between two groups of PAMs is obviously different under four challenge doses of foreign PRRSV highly pathogenic strains NVSL 97-7895.

4. Determination of viral titer by TCID50 method

a. Selecting 1 PAMs which grow vigorously after 24-48 h culture, subculturing into 96-well cell culture plate, and counting with cell counting plate before plating to make about 2 × 10 cells in each well of 96-well plate⁴And (4) respectively. Then culturing in a cell culture box for 12-24 h, and preparing for virus inoculation when the cells grow to 80-90% confluence;

b. 8 1.5 mL centrifuge tubes were prepared, 900. mu.L DMEM maintenance medium (containing 2% FBS and 1% diabody) was added to each tube, and 100. mu.L of virus stock (foreign PRRSV highly pathogenic strain typEII: NVSL 97-7895) was diluted 10-fold in a gradient (from 10 to 10)^-1Diluting to 10^-8 )；

c. The culture medium in the 96-well plate was aspirated, washed once with PBS, and then inoculated. Starting with the highest dilution of virus solution, virus was inoculated from high to low, 8 wells were inoculated for each gradient of virus. Meanwhile, only adding a maintenance culture medium into cells with at least 5 holes, and not adding virus liquid to serve as negative control of no virus infection;

d. culturing the culture plate in a 37 ℃ and 5% CO2 incubator, observing the pathological state of the cells every day and recording, wherein the process generally needs 4-5 days;

e. after 4-5 days, the number of wells for each gradient of cytopathic effect was counted and TCID50 was calculated.

The results are shown in FIG. 7, the virus titers of the foreign PRRSV highly pathogenic strain NVSL97-7895 in the supernatants of the two groups of PAMs reach very significant difference (P < 0.001) under four challenge doses. The results show that the CD163 gene modified porcine PAMs have strong resistance to PRRSV virus under different challenge doses.

Therefore, the method proves that the CD163 double allele knockout pig obtained by the invention has the capability of resisting the PRRSV virus, and the method for preparing the CD163 double allele knockout pig can obtain the PRRSV virus resisting pig, the obtained CD163 double allele knockout pig can be used as a disease resisting pig for propagation, the somatic cell nucleus of the ear sample of the CD163 double allele knockout pig can be used for propagation of the disease resisting pig by somatic cell nuclear transplantation, and the CD163 double allele pig fibroblast mutant can be used for propagation of the disease resisting pig by somatic cell nuclear transplantation.

SEQUENCE LISTING

<110> Beijing first agricultural future Biotechnology Ltd

<120> method for preparing CD163 gene editing pig by using SpRY-Cas9 system

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 32457

<212> DNA

<213> Artificial sequence

<400> 1

attcttagtg gttctcttct tcaggagaac atttctaggt aataatacaa gaagatttaa 60

atggcataaa accttggaat ggacaaactc agaatggtgc tacatgaaaa ctctggatct 120

gcaggtaaaa tcttctcatt tattctatat ttacctttta atagagtgta gcaatattcc 180

gacagtcaat caatctgatt taatagtgat tggcatctgg agaagaagta acagggaaaa 240

ggcaataagc ttataagggg aacttttatc ttccatagaa tcaaaattga agacgtgact 300

agaagaagga ttagatttgg catcagtttt gtaaaattgc tgaggtgaaa ttaagtaagg 360

gatgaaaatt aactaaattg tgttgagtat gaaactagta gttgttagaa aagatagaac 420

atgaaggaat gaatattgat tgaaagttga tgacctagag gacatttaga ctaacacctc 480

tgagtgtcaa agtctaattt atgatttaca tcgatgcgtt aaactcattt aacattctta 540

cttttttccc ctcaagcatt taagctgaag tataacattt cacatgaaag cctggattat 600

aaatgcacag ttcagtgacc tatctcagag gagtgactgc catagcattt tttttgtctt 660

tttgccttca gagccacagc aacgcgggat ccgaagccgc gtctgcgacc cacaccacag 720

ctcacggcaa tgccggatct ttaacccact gagcgaggcc ggggatcgaa cccgcagtct 780

catggttcct agtaggattc gttaaccact gcgccacgac gggaactcct accatagcat 840

ttttactttt aagttactgt tggtttagag taagaaggag aaatgagagt gatggagcgt 900

ttgctatatt tggagacaag gtcctatatt ggaggttctc aaatataaat tttgtcgctt 960

tttcctccaa tgtattgttc aactactatt tagcaggcca ctgtgccagg tactggtgaa 1020

actggtgaac atgatagatg taattcattc cctcatggaa ctttccatct aacaatgtgg 1080

atcaggtagg cttggagatg agaatgccag tggttgacta tgactctgtg gctgaaggga 1140

gagctactca cttcgtagtt tcatcaatgt ctttttggtt ttccaggttt taagccctgc 1200

tcttgcaatt cttttccctt ctccaacttt cttctaattt ctcaccccta ggatgcctat 1260

aaacatgagt attttcaaag ctacttcact gaggttatat gatcctcgtg tgaatttttc 1320

ctgcctgcct tgccatttag aaggaagtgt ttcctggaat ttccattgtg gcttggtggt 1380

taaagaccct gcattgtctc tgtgaggatg tgggttcaat ctctggcctc attcagtgag 1440

tgggttaagg atctggtgtc gctgcaagct gtggctaaga tcccacattg ccatggctgt 1500

ggtgtagact ggcacctgga gctctgattt gaccacaatc ctaggaactt cagatgttgc 1560

cataaaaaga aaaaaaaagt taggaagggt tttctgtctt gttttgacct ttgttaatct 1620

caaacctttg gaaccatctc tcctccaaaa cctcctttgg gtaagactgt atgtttgccc 1680

tctctcttct tttcgcagac tttagaagat gttctgccca tttaagttcc ttcacttttg 1740

ctgtagtcgc tgttctcagt gcctgcttgg tcactagttc tcttggtgag tactttgaca 1800

aatttacttg taacctagcc cactgtgaca agaaacactg aaaagcaaat aattctcctg 1860

aagtctagat agcatctaaa aacatgcttc atggtttcaa aggatcagat attaaaaacc 1920

ccaaataggt acagaaccat gtggctctct ccccccaaac aaataaaacg ttagcatggt 1980

tttcaaaaaa ataaaataac cttcacagga aaaatggatt ttacttaaga tttgaaataa 2040

tatctaacta aaaaataggg aataatgcag aagaggagaa acctcagaat tgttgggatg 2100

aaggaatttt tagtaacact aaaaattcaa gtgccaaaat ttgtctaaaa ttgtattcag 2160

ggaagccaga tatatatcag tgaaatcgcc agttcctata ttagctaaaa taatcacaag 2220

gctgtagcag agacagttca gagagaggtg gagatgagat tttttttttt taagtataat 2280

tgatttacaa tgttgtggca atttctgttg tatagcaaga gatagaatta ttttatggtg 2340

gaagataata gaaaaatata tccatatcaa tttccatttg agtagataaa tttcaattag 2400

agttcaacta gcaattagta gttttgcata catggtgaaa tatattcatg gtattttgca 2460

tatatgtgtg aaataggtac taaattcctc ataactgttc tttttagtct caccatcagc 2520

ctctactgat cttaggattt tggagaaaca tacatagttc atccctataa aatgccataa 2580

aatctcattt ttacattaaa ccatccaaga gattatataa attgacctta taaagaatat 2640

cagccataaa ataaaggtat catagtatgg gattatttag ctttattggt tctatgtcac 2700

tgcttaattt gaaacctgtg atattgctgt ttgtttttga actcctatga aataacattc 2760

tcccattgta ccatggatgg gtccagaaac atttctcaaa tctggctttg aaaaataaat 2820

aagtaatcta aagaataata attctctact tgctctttga atcttgacca attgctgcat 2880

ttacctattg ttacaggagg aaaagacaag gagctgaggc taacgggtgg tgaaaacaag 2940

tgctctggaa gagtggaggt gaaagtgcag gaggagtggg gaactgtgtg taataatggc 3000

tgggacatgg atgtggtctc tgttgtttgt aggcagctgg gatgtccaac tgctatcaaa 3060

gccactggat gggctaattt tagtgcaggt tctggacgca tttggatgga tcatgtttct 3120

tgtcgaggga atgagtcagc tctctgggac tgcaaacatg atggatgggg aaagcataac 3180

tgtactcacc aacaggatgc tggagtaacc tgctcaggta agacatacac aaataagtca 3240

agcctataca tgaaatgctt tgtgggaaaa aatgtataga tgagttaaaa acaaaaagga 3300

accagttttc tataagtcat ctagtccatg tataaaatta cccaatccat tactaaaaga 3360

ccacttctgg tattttacac atgacaaagc ccatattaaa aaaaaaaaat tcagaagaga 3420

ttctgaatgc tataataaat gagcaagtga ctagcttcaa ttttatatta ggtcattcta 3480

ccttctactt ctacatgaaa atatcataat gtctaagtta attccttgtc ccctttccca 3540

ataaagcact gctttcatgc actggcctat gaatcatgaa ctttttgccc tttaactgat 3600

gatcaactta ccaaatcaag aaataaatat tcttagcact gatccttttt tgttgttgtt 3660

ggaggaagaa tgttttgcaa agtagaattg cttttttctg tttaacagtg ctattcattt 3720

catttacatg gtcgttttaa tttataaaac atttcataag tttcacctca tatgccctta 3780

caataactca ggaagttata tgttagacct ttctgctgac aaatcccaga gtcatgtttc 3840

tgacccagtt cagattcctt ggcttcccat ttctctttgc tcatgtcatt gacctttatg 3900

cagccctctt acctcccacc tttctattac agaccatctc ctccatagga ctggtgttag 3960

aaagtactaa tctctaccca ggcattgtgg tgcaatgtgg gcagcacagg ctggtatcta 4020

gaaaaatgct gaagtgaatt ccagctcagc tgctcgttaa tactattgtt ttaagtaagc 4080

tgttcaatcc tttgaaattc actttctgag cactcagtga tataataaat gtagagttac 4140

tggtacactg tctggtatgt aataggtgtt aaaaattaac cttagtttcc tcatgggtca 4200

ctgcttctca ttacctagac aactcatttc tctttcttcc tctttctctt tctccattct 4260

cctcctcctt cttcctcttc ttcttgtctt ttattgttat tcattttgct gagaaagtta 4320

agaaataaca actctaacct ctacatcgac cacctagagc aaagttaaaa ataataataa 4380

accttgccag actcttacta taattgttgc tgtctataga gttgactgtt taagttaaga 4440

catcagtata gtatttttaa tttttgtgtt ttttttttca tacttttaca tgaggatcct 4500

ttatataagg atgagttaaa caaacttgat ttttgaagtt tatacccctg aggctcaact 4560

gcataataat agaaagggat ccatagcctc tcaaggactt aactagtttc atgagttttc 4620

agaatctgaa tttctgagat tctccacccc aattaaagct caagcctcag aacatatatc 4680

cttctcttgg taaattctat tcttatcaca tgcgtaataa taaaaaagag agatgttgga 4740

gacagatttt tttcctcaca ttctgtctct actgttttct aggtgtttga ttctgtgtta 4800

tttaacctca gtttgcttat ctgtgaagta gggattatgg taataacata taatgcttaa 4860

tgttgtaaag actaaagaag atagcatatg taacacattt ggaacaggga atgcatattt 4920

tgattgtgag ctcttattat tattaccaat cagccataat aaaaatcttg ttaagtggag 4980

gtctttggat ttcagagctt ttaaaatcta attacttttt caaaaaagag cttcttagtg 5040

tttttttttt ttaaccacaa agtgtttcta ttttttaggt gtcccaaaat ttcattccaa 5100

atatcttttt ctcagatatt ttagtcctca tagaacacct agggatagtg tatagagaaa 5160

attttcttta ttaaaaagct gttctttgct aaaaattgta gcaggtactt ttgggagggg 5220

ggaaaacttt gattcagaaa ctgctaagac atggagtgtt ttgactaatt tttcctcaat 5280

ttttaatgtt ttttatacca tagggtactt ttgcaaacta ttatgcatac ttatatattt 5340

ttactttttt cctgtctttt aacttccaaa ttcaacttca gacaattatt catgcactaa 5400

actgttgtag taagaaagat taaaattaaa aaattaacca ttcaacaaat gactggtttg 5460

ccatttttac tactttgttg tatgaacaat ttttttttct acaaatgaat actttgagtc 5520

tgatttatcc attcctacat aaaagttttt actatatctt agtattggaa ggaaacaaaa 5580

caaaacacaa tgtaaatttt aatctataaa ttttgggggg gggtaaatat acatagatga 5640

aagtcttaac cattaattag agtcaaaaga ttaaaattct ccaatatgtg aacttaggct 5700

gcatccaaaa tgaagcatca tttttaagga cagcatcaaa agtgaccaga ggaattttac 5760

tttctttctt tttttttttt ttttgaattt tagtttctaa actcacttct gaataaatac 5820

aacttctaaa ttctcgtctt ttctctactc tagatggatc tgatttagag atgaggctgg 5880

tgaatggagg aaaccggtgc ttaggaagaa tagaagtcaa atttcaagga cggtggggaa 5940

cagtgtgtga tgataacttc aacataaatc atgcttctgt ggtttgtaaa caacttgaat 6000

gtggaagtgc tgtcagtttc tctggttcag ctaattttgg agaaggttct ggaccaatct 6060

ggtttgatga tcttgtatgc aatggaaatg agtcagctct ctggaactgc aaacatgaag 6120

gatggggaaa gcacaattgc gatcatgctg aggatgctgg agtgatttgc ttaagtaagg 6180

actgacctgg gtttgttctg ttctccatga gagggcaaaa aaaggggagt aaaagtctta 6240

aaagctcaaa ctgttaaaaa cataatgatg attgcttctt ttatcatctt attattatct 6300

aatttcaggt cgaaattcta gtacctgtgc agttttttac cttaactgaa attaagataa 6360

ataggatagg gaggaaggat gagcagtgac atttaggtcc aagtcatgag gttagaagga 6420

aatgttcaga gaatagccca ttccctcagc cctcaaagaa agaaagaaag aaaaagaaaa 6480

aaaaaaagaa agcttaacta gaaaattttg ttctctggat gttttagagg caaaccatcc 6540

cttttatcat tccttaccta caaagccctt ctctttaatc acattgaccc accctttcct 6600

aaactattag ttcaaattca cataattgaa tgcttttaaa acttggtttc ctcttataat 6660

tatatttatg ttgtaaggag gcactgtgtc ttgtctagag actttcatgt tctatgcttg 6720

attatgggac agggacatgg ctttgtctgc tccaggatgt cactctcctt ttttcacttg 6780

agctcctagt ttgaagaaga cctagtaagt cttgaactcc agggagtctt taggaaacta 6840

tccctagagc aaaactgtcc ctgaattcac ccagtgtctt tttttttttt ttcaaatgaa 6900

ggaactttag ttcaaactaa atttaaaata agggaattct aattcagaat actgggaaat 6960

ccaggagatt acaattggct tcatgtgtga ttggattcag cacttcacca atgtcatcag 7020

ggttctggtt ctttttttat ttcttgaatt ggcttttttt tttttttcct tgttgaacaa 7080

tatgactatc tatactttga accacaaaga aagtgattcc tacagaaaag acagaatgtg 7140

ttagctgaag gaagggaatg ggacttgggg tagaaaaaaa caccttccgt attccttaac 7200

ctatcaaaaa tttctaggta cccctaacta aaatcctaat tcaagcatat tggaggaact 7260

tgacaaatcc aggaataata ttatccgtta tcaaatacat gcacatcatt tacatttctc 7320

catgtctctg ctcatgcagt tcccggccct aactctacca aagtattact ctccatctcc 7380

ctcttttttt ttttaatgat ttttattttt tctgttatga ctggtttaca gtgttctgtc 7440

aattttctac tgtacagcaa agtgacccag tcacacattc atatatacat tctttttctc 7500

acattatcct ccatcaggct ccatcacaag tgactagaca tagttcccag agctatgcag 7560

caggatctca ttgctgctcc attccaaagg caacagttca catctattaa ccccagattc 7620

ccagtccacc ccactccctt cccctccctc ttggcaacca caagtctgtt ctccaagttc 7680

atgagtttat tttctgtgga aagttttatt tgtgcagtat gttagattcc agatataagt 7740

gctatcatat ggtatttgtc cttctctttc tgactgactt cacaaagtat gagagtctct 7800

agttccatcc atgttactgc aaatggcatt attaatctcc atcttttttt gttcatgtat 7860

atgttaccca gattccttga cttttctaca tcatcaagat attgttgatc acttctttgt 7920

agtgatttct gcccttctct gatgtcctgt gacactagtc tggattattc atttacctga 7980

aaccacatgt ctcttataat gtgtatccca aattaaatat gtctattgta atgtgtatcc 8040

caaattaaat atttatcttt ctaaaaaaaa aaatttctag gcccccaatc agcatgtttc 8100

ttctcagtgt gttttataca tgctgcagaa tcataataga cagcataata gacagcataa 8160

caaaaactaa aaatgccagg ggaaaaaagc aatttactga ttacaacata ttactcagaa 8220

tcaagttctg ttctttgagg aatattgatt gggggaaaat gaaaataatg atggggaggt 8280

cccttttctc tttgctttgc ttttaaacta cggaagtagt cagaaagggg tcaggaatgt 8340

aatataaacc aggtagtcct ggtaggtaac gcagccggag gcaaaagtga gtgttgagta 8400

ttgaggcaaa ctggagggca tggataccac ctagacagat gcaaatatat atttaacagg 8460

gaaaaaagaa ccaaacaatt tcaacaaaaa accaaacaat tccaacaaaa ttggtccaat 8520

aagcaaacct ctagataaat ttcagtccct ggatgttttg ttaggaactc ttcctacaat 8580

gcgtgctttc cattctgaaa agtcctatct acttgcctga tccacttctc cttccatcct 8640

aaacgatttt cagtggtagt atattactgt tgtctctgtc tctacttata tatcttcccc 8700

ttttcactca ctcctctcag gtacagctct tcagtttgcc cttattcttg tttccttgtc 8760

aatgacttgt tttgtgtccc tcttacagat ggagcagacc tgaaactgag agtggtagat 8820

ggagtcactg aatgttcagg aagattggaa gtgaaattcc aaggagaatg gggaacaatc 8880

tgtgatgatg gctgggatag tgatgatgcc gctgtggcat gtaagcaact gggatgtcca 8940

actgctgtca ctgccattgg tcgagttaac gccagtgagg gaactggaca catttggctt 9000

gacagtgttt cttgccatgg acacgagtct gctctctggc agtgtagaca ccatgaatgg 9060

ggaaagcatt attgcaatca taatgaagat gctggtgtga catgttctgg taagtgaaaa 9120

caaaacaccg gaaggacctg tgttcttcag gattaggaat ggatatgaga taggagaaaa 9180

attgtatcta atattttctt tgttgggaat tcttttacag ttgtgacaaa tctttaacat 9240

attcttcatt tgagtagttt ggagggttgt ctgactgttt tctataataa atgtcccaag 9300

tgctatgagg taccacattt caaattctaa ttctacctga agctccaaaa agacaaaatg 9360

ttataggtct tttctttata tctaatttgc ttatggtttt tagccattga caattttttt 9420

tttcttaact cttgaaacta taatcctatt tctaaccaaa ttcatgttct atactggctc 9480

ttcaaaaacc caggagatgg gaaagccaga atctccagtg tttcagcttc tgggaaggag 9540

caagttttta aatgtgggag ctaaattcca catgtatcta tggcctaagt gtatgtttat 9600

tttgcagatg gatcagatct ggaactgaga cttaaaggtg gaggcagcca ctgtgctggg 9660

acagtggagg tggaaattca gaaactggta ggaaaagtgt gtgatagaag ctggggactg 9720

aaagaagctg atgtggtttg caggcagctg ggatgtggat ctgcactcaa aacatcatat 9780

caagtttatt ccaaaaccaa ggcaacaaac acatggctgt ttgtaagcag ctgtaatgga 9840

aatgaaactt ctctttggga ctgcaagaat tggcagtggg gtggacttag ttgtgatcac 9900

tatgacgaag ccaaaattac ctgctcaggt aagaatttca atcaatgtgt taggaaattg 9960

cattctactt tcttttacat gtagctgtcc agttttccca gcaccacttg ttgaagagac 10020

tgtcttttct tcatcatata gtcctacatc ctttgtcata aattaattga ccataggtgt 10080

gtgggtttat atctgggctc tctattctgt tcctttgatc tatgtgtctg tttttatgcc 10140

agcaccatgc tgttttgatt actatagctt tgtagtatca tctgaagtca ggaaacatga 10200

ttcctccagc tttgttcttc tttctcaaga ttgttttgtc tattcagagt tttatgttcc 10260

tatgcagatt ttatttttat ttttatttta tttttatttt ttttattttc ccactgtacg 10320

gcaagggggt caggttatcc ttacatgtat acattacaat tacagttttt cccccaccct 10380

ttcttctgtt gcaacatcaa gggcagggac cgaacccgca acctcatggt tcctagtcgg 10440

attcgttaac cactgcgcca tgacgggaac tccctattat ttttattttc taatttgttc 10500

atgtggtgta tcacactgat ttatttgcag atgtgcatcc attcatgtat cccacttgat 10560

cgtggtgtgt aatcttttta gtgtattagt gaatttggtt gctagtattt tgtttgagga 10620

tttttgcata tacattcatc agcggtattg gattttaaat cttttgtatg tgtcttgttt 10680

tggtatcagg gtatcctcta gggtatcctc ctagaatgag ttcagaaggg tacatttctt 10740

tggggaatat atttggtaga attcactttt gaagctgtct ggtcctgttc ttttgtttgt 10800

cgggaagttc tttttaaatt attattatta ctgattcaat ttcattactg gtaattggac 10860

catttatatt ttcttttttt tcctggttca atcttgggag attgtatgtt ttaaaaattt 10920

gtccagttct tctaggttgt tcattttatt ggaatgtaat tgtttgttta tctttttttt 10980

tgcattttct agggccgcac ccatggcata tggaagttcc caggctaggg gtctaatcgg 11040

aactgtagcc actggcctac cccagagcca cagcaacgtg ggatctgagc cgcatcttcg 11100

acctatacca cagctcacaa caatgcggga tccttaaccc actgagcaag gccagggatt 11160

gaacctgcaa cctcatggtt cctagttgga ttagttaacc actgagccac gacgggaact 11220

ccaatggtat gtaattgttt atagtgatct cttatgagtc tttatttttc tgtagtaatc 11280

ataacttctc ttatttcatt ttgatcttat tgacttgagc cctctgtttt tttcttagtg 11340

actctagcta aaggtttatc aattttgttc atttttttca aggatctggc tcttaatttc 11400

attcaacttt tctatttatt ttagtctcta tttcatttac ttctgttcag atttttatga 11460

tttctttctt tctactaagt tcagttttgg tttgttcttt tctatttcct ttaagtgtaa 11520

ggttatgttg tttatttgag atttttgttt cttgaggaaa caggcttgca tatttgtaaa 11580

cttccctctt agaatagttt ttcttaagtt ccatagtttt ttttttttat tttgtggttt 11640

ttatttttcc attatagttc atttacagtg ttctgccaat tcctactata tagcaaagtg 11700

acccagtcat atatatatat atatatatat atatatatat atgtatatat gtatatatac 11760

acatacatat acacattatc ctccatcatg ttccatcaca agtgactgga tacagttccc 11820

tgtgctatat agcaggatct cattgcttat ccactccaaa tgtaatagtt tgcatctatt 11880

aaccccagat gtcccataga tttggaattg tgtttttgtt ttcattcgta ttcaggtttt 11940

ttttaatttc ctctttgatt tcttcagtaa tccatttgtt gcttagtaat atattgttta 12000

gcctctgcgt gtttgtggtt tgttgcaatt ttcttcttgt agttgatttc tagtctcttt 12060

gtgttgtagt tggaaaagat gtatgatatg atttcaactt tcctaaattt accaaggctt 12120

gttttgtggc ctagcatgtg atatatcctg aagaatgttc catgtgcaca tgaaaaaaat 12180

gaatattctg ctgctttcaa atggaatgct ctctctattt caattatgtc catctctaat 12240

gttttgggaa catgttcttt tgctacctca ttttgcctaa tttgctgttt tgggttctaa 12300

atatctggta ggttggttac attttccaac cttggacaaa taaccttttg ttgaaacatc 12360

ctgtgcttcc cagcagcact ctcctctctg gtcaccagag ctatatgttc caggggtgcc 12420

cccctatgct gactttgtga gaacttcttt tgcagttggc tgactactgt aggtggtctt 12480

gtaggcatgg ctggccccca gtctggttgt ttgcaagaag ctgccttgta caaaggctgc 12540

cagtcacttg ttggtgggac tgggtcatgg ggtggctggc tatagagacc agggttgtct 12600

caggggtagt gctgtctcat ttgtgggttt agccacgttt tgcagtgggt gattgtggtt 12660

ccagggttcc tagatctagt gtcagcttgt gggtactggg gtccccagct gcagggccta 12720

ggagcttcag agctagagct aacctcctgg tgggtagact gtgtcctgac aaggcaggtt 12780

gtagtgttac agtgatcctg gggctagtat ctatccactg gggggtaaga cttgtcccag 12840

ggctagcacc agctctctgg tgggtagatc taggtcctgg aggttctggc tgcagggcca 12900

gggatccagg agctggtgtt gactggttgg tggacagggc caaggcccag agtgtcccca 12960

ggctagatct acttcagtga tgggtggatc taggtcctgt atttctggct acagggctct 13020

gggatcccag agttggtatg tcagtcaact gacatacagg gctggaggca gagagtcctg 13080

aggctggtgc ctgcccactg gtgggtggag ctgggattca gggtctctga ctgaagtgcc 13140

ctggggatcc ctgggctagt gctggcccac tggtgtgtgt ttggttgggt cctggccatt 13200

ctggtagaca gggccatatt cccatattcc agggtggctg taggctcagg gaatctcaag 13260

gcaacctact gctggttaga ggagtgtgtg gggaggtgct atgtccctgt ccagtttgtt 13320

gcttggcatg aagcatccca gtactggtgc caacaggcta attagtgggt ctgggtcctg 13380

gtgctaataa gctagaggga agattcaaaa atgacatttt tttaacacca gtgtccttgt 13440

ggtaaaatga actccccaga atggctacca ccagtgtcta tgtccccatg gtgaattcta 13500

attgctcctg tctcttgaag tggctctcca agatcaacag gtgggtctga tctaagctcc 13560

tttcaaatta ctgcttctgc cctgggtccc agaacatgtg agattttgtg tgtcctttaa 13620

gagtggagtc tctatttccc actgctctct ggttctcccc aaagtaagcc ctgctggctt 13680

tcaaaacttc tgggagcttg ccttcttggt ataggactcc tgggctaggg agtctaatgt 13740

ttggcttaga ccccttactg cttgggaaga atctctgcaa ctgtaatgaa ttatcttcct 13800

atttgtgggt tgctgaggat atggtcttaa ctgttctgtg ttctacccct cctatccatc 13860

ttgttgtggt tccttcttta tatctttagt tgtagaaaag tttttcttat caacagttgc 13920

tctgtaaatt gtaacttggg tgtacaccta gtaggaggtg agctcagggt cttcctactc 13980

tgccatcttg gccatgtcct ctaaacattt tggtgtattt cactgcaacc tttttaaaaa 14040

tctcaaaagt gagctgtgat tggctagtct tgtggataat ctctagcatt tgatgctaat 14100

catatttata caaatacttt gttgaaaagt gatgcctttt taactattat taaaaaacgt 14160

attgacataa ctattgctat tatactgaaa agaaagacct tagagaaaat agcataagag 14220

caaaaccatt aaacatggag acatctagtc atagggtgga aattttatgt ggtgcatatc 14280

ccctaaccag tggctttaca ccaggcacat cctaactaag atctgctccc aagtgtcttc 14340

cctgatgctt taaattgtgt tacatggaaa ctatcctttg atgaagaaat gcaacctttt 14400

aaaatacaac attgaaactt ttgtgcttta attttgcttt tcaacatttt ttctttttaa 14460

aagaagaaat ttatttgttt ttttaaattt taatggccac ggcatatgga agttctcagg 14520

ccagggatag aattcaagcc acaggtgcga cccatgccac aactgctgca acaccagatc 14580

ctttaaccca ctgcaccagg ccagggattg aagccttgcc ttactgacaa tctgagccac 14640

ttcagtcaga taaagaaatt tcttcattaa gcagagtatt cacatggttt aaacttcaaa 14700

atattaaagt gtaaactctt tccccaccac tgtccccagc tcaccaactc tacttaccac 14760

agacaactga tgtggttagg gtatttaaat agtaaatcca agaaaatata aacaaatccg 14820

tatatatagg tttcacccca ttttattatc ctaatgttgc atatcatata aactatactg 14880

tcccttgggt attcacttag taaaatattt tgatcataat ttcctatcag tatttaaaga 14940

gctttctgaa attatttctg tataacattt cttttctcat catctattat gtgcatttat 15000

ttatatttta acttctttta ttagatgaaa ttatcttctg cttcagcttt tttttttttt 15060

taagaacaca cagttgggtt ttttaaggtt aataccacct ttgttttcta agtcattaaa 15120

tttgtttttc tattaattca cttctgattc tttgaagttt gatttctttt tagcttttaa 15180

cttcttgagt tgtatgctta attaattttg attctttcct atttattaat atacatattt 15240

gaagctatag gttttccact gagtatacca gtagctatat cgtataattg atgaactgat 15300

cctctgtgag tctgggacat aaacgtccta tgactgttat gtggtagctg tgaattgctc 15360

tttttagatt ataaagttct catcttttat agttgaacaa tttttgtcct gaatcaaatt 15420

tgttggatat taatatcaca tctattgctt tatttatttt ctattctcac ttttaacctc 15480

tgtgaataat ttcactctag gtgcctcact tttttcataa tagaattggg atttattttt 15540

aaaaggactc tgattaagta attttctttt tctgatatgg gagatatatt tgaccttaac 15600

ttagtcacat tatgcattgt tctcttgtca tgttatgtat acataacatt tattgtcatt 15660

atggtacaac taaaaacata tttcactctg tgacctttat ggggactcag catttgttta 15720

ggaatgtgga agtatatttg tatatctgat aatttccttc caaatttaaa aaggtttgta 15780

tattttcata ttaacatatt tcatattaat tagcatgaat ttcagctgca ttaaaaggaa 15840

aaccacctga gtggtaaaga aaaagttttt ttttctcttt tttttttttt tttttttaat 15900

ggccacatct gtggcatgtg aagttcccag gctaggggtc gaataggagc tacagctgcc 15960

agcttgcacc acagccacaa caatgccaga gccaagcctc atctgcgacc tataccacaa 16020

ctcatggcaa tgctggttcc ttaaccccct gagtgaggcc tggggtcaaa cccacatcct 16080

catggatact aaccggcttt gttaccgctg agccatgagg gaaactccct ttttctcatt 16140

gaaaataagt caaatagata agcagcttaa ggctgtttgg gtgattctgt ggtccagtaa 16200

ttatcaaatc ctactggaca agaatagaga atgtgcaaat gagggaacgt gttggtgaga 16260

tcaggctctg cccactgagc tatcctctgt catgggccct gtgctgttct cagagctgta 16320

cttcctaggg cattgttctc atttcaattc tgagttcagt gtggagagta tacgtgtgtg 16380

ggggctgcac gcttttcaca acccactttc tgctgatact gatttaggga tccttggatt 16440

gctttacagt tgagtcatca ttaactagtg tcacttgcct tcaaagtcag caaaataatt 16500

gtctccaaac tagtaggctt ctagtgtatt tgctttaatc caatgccatg tgaaagtaac 16560

atggtcaaag aataagttat ataccttgac ctaccctgtg accaggctct tcctcttaat 16620

ttattgacca ctgccttaag gtcatttgaa accatgggtt tgggaggaag gcaaggccta 16680

aatcccgtct ttgttggaag gctcactgtc cttgtcttta gagcatcatt tttttttaaa 16740

ctggggtaca gtttatttac agtgttgtgt caatttctgc tgtacagcat agtgacccag 16800

tcatacacat acatacattc tttttctcat actatcttca attttatttt gtgctaagtc 16860

tgccatttta tcatcacctc agtttgaagg acaggatatt tagagtttgt tttttttttc 16920

cccccaatcc tgcaatttct aaattataag actctcaatt agccgtatat aacagctgca 16980

ggcacaggat gtctccctca caaaattggt atttttcctt ccatttcttc ttgcagtttg 17040

gctatttctt gtctgagttc atctctcttt ttaagtgtta aaaagggcaa ggaggattca 17100

tgctatgtca acattatgat tttttctttt ctatacttga taagagtata cttttcccaa 17160

atgtcatcca acttttcagc atcagtttgg acatggtttt cttttcaagg tggtatttct 17220

ctaatgtcac ttgaataaca agactcgtta gttctccagg ctacaatatc ctagtctgag 17280

tatattctgc atgttaattc tattcagcca catccataat ttaggtttta ttcctggaac 17340

acctcacttt tttttttttt tttggtcttt ttatagccat aaccatggca tatggaggtt 17400

cccaggctag gggtctaatc tgagctttag ccactggccc atgccacagc cacagccatg 17460

ccacatctga gccacatctg tgaccttttc cacagctcac agaaacacca gatccctaac 17520

ccactgagtg aggccagggg tcaaacctgt aacctcatgg ttcctagtca gattcgtttc 17580

ctctgtacca cgatgggaat tcctaatacc tcacttatga taacacattc tgaattattt 17640

aggattctat tatactgcat gtaatagaaa tcccaaatag caaaatttgc aacttaaggc 17700

aggttcctgt ctttacaaaa tcatgttttc ctttgctata tgtgcacttt gctttcctct 17760

gtgaattccc ttttttgtta tatttctata gcttttggaa acacttttac ttatttgggg 17820

gggcctagat ttttaaccct ctccttgttt ttctagaaat agagtttata attttatttc 17880

ttcatttact tgatactttc aagagatttc caggaaaaaa attatggaaa tactgtctct 17940

gtgcctgcca agttcaaact aagaattgta taatctgttt taattcttaa gcatttatag 18000

atgacaaggc tttgtgtctg ataggggcca gcgaactcag taaagaggga agatgagaaa 18060

gataatggca agaatttatc cctgaagtgt agttttgaca aaccagtcac aaagaggtct 18120

aagaaatttt ggtcacaaag ttgttttgaa tcccaggcat tttatttgca atgattgcat 18180

atgttctgga aaggacatct gaacctaaga aatagttcat ttgcattgtg ttatatttta 18240

ctaaggtctg agaaataatc ttgagatgag aatgaactct acttcttcag agtctggaag 18300

gaataaatta tgaaaatgta ttaatgcttc tttaaaccat attgtatatt tatctattac 18360

taaacaaaaa gaagtagctc tatttattta tttatttatt tatttattta tgtcttttgt 18420

ctctttaggg ccacacctgt ggcatatgga ggttcccagg ctagaggtcc aattggagat 18480

gtagcagcca gcctatgcca gagccaccgc aacacgggat ctgagccacg tctgtgactt 18540

acaccacagc tcacagcaac gcctgatcct caacccactg agcgaggcca gggatcgaac 18600

ccatgtcctc atggatgcta gttgggttca ttaactgctg agccatgatg ggaactccaa 18660

attaattatt tcttatattt gttcttcata tattcatttc tatagaaaga aataaataca 18720

gattcagtta atgatggcag gtaaaagctt aacttattaa tcaaaggagt taatccaggc 18780

acaaaaattc aattcatggc tctctgttaa aatttaggta taggtttagc aggaagaaaa 18840

ggttagtaga tgcagactat tacatttaga atggatggac aatgaagtcc tactatacag 18900

cacagggaac tatatccaat ctcttgggat agaatatgat ggaagacaaa atcagaacaa 18960

gagagtatat atatatgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 19020

gactgggtca ccctgcggca cagcagaaat tggcagaaca ttgtaaatca actatacttt 19080

aataggaaaa atacttttaa gggctaaatt tccaatattc taaccatgta cacagagtaa 19140

atgtcataag gatgccagtc tgtgtagaga ttgatgtgtt actagcagat tcatgaaata 19200

aaggctgagg atgtagtccc caagtcactt ctgagtggaa gaatttctcc tttgtcctgg 19260

actcaaatat tttaggataa aggaaaaaag aagatattta tagaagggac ttgttttcaa 19320

gtacttgaca aaatttcacc attaaagaga aatttgtggg agttcccatc gtggctcagt 19380

ggaaacaaat ccaactagga accatgaggt tgtgggtttg atccctggcc tcactcagtg 19440

ggttaaggat ccggtgttgc cgtgagctgt ggtgtaggtt gcagacacgg ttctgatcct 19500

gcgttgctgt ggctgtggct gtggtgtagg ccagcagcaa acagctctga ttagacccct 19560

agcctggaaa cctccatatg ccacaggtgc agccctaaaa agacaaaaaa agagaaaaga 19620

caaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaga 19680

accaccagag gtatttattt gtttttgcct tttttcactg actgtttttt gtttgtttgt 19740

ttgagactga tctagaagac tagagattac aagaaatatg gatttggctc actctaagaa 19800

actgctttca ttccaaggtt tgggtctatc caaaagtgga atagaatcat atgaatacta 19860

gtttatgagt atttagtgag aggaatttca agctcaaata atgattcagc aagattaaat 19920

taaggaggga attttccttg tggctgagtg ggttaaggac ccaatgttgt ctctgtgagg 19980

atgtaggttc catcctgggc tttgctcatt aggttaagga tctggcattg ctgcagctca 20040

gacccagtgc tgccctggtt gtggcttagg ccaaagctgc agctccaatt caatctctgg 20100

cctgggaacc tccatgtgct acaaggtgcg gccttaaaag gaaaaaaaaa aaaattaaat 20160

caaggactca agagtctttc attatttgtg ttgtggaagc tatatttgtt ttaaagtctt 20220

agttgtgttt agaaagcaag atgttcttca actcaaattt gggagggaac ttgtttcata 20280

catttttaat ggataagtgg caaaattttc atgctgaggt gatctatagt gttgtaatgc 20340

agaatatagt cagatcttga acattttagg aagttggtga gggccaattg tgtatctgtg 20400

ccatgctgat aagaatgtca agggatcaca agaattcgtg ttatttgaca gcagtcatct 20460

ttaaaaggca tttgagaaag tccaatttca aatgcatttc ctttctttaa aagataaatt 20520

gaagaaaata agtctttatt tcccaagtaa attgaattgc ctctcagtct gttaaaagaa 20580

actcttacct tgatgattgc gctcttaacc tggcaaagat tgtctttaaa atctgagctc 20640

catgtcttct gctttatttc tggtgtgcct ttgactccag attacagtaa atggaggact 20700

gagtataggg ctaaaaagta gagagaatgg atgcatatta tctgtggtct ccaatgtgat 20760

gaatgaagta ggcaaatact caaaggaaag agaaagcatg ctccaagaat tatgggttcc 20820

agaaggcaaa gtcccagaat tgtctccagg gaaggacagg gaggtctaga atcggctaag 20880

cccactgtag gcagaaaaac caagaggcat gaatggcttc cctttctcac ttttcactct 20940

ctggcttact cctatcatga aggaaaatat tggaatcata ttctccctca ccgaaatgct 21000

atttttcagc ccacaggaaa cccaggctgg ttggagggga cattccctgc tctggtcgtg 21060

ttgaagtaca acatggagac acgtggggca ccgtctgtga ttctgacttc tctctggagg 21120

cggccagcgt gctgtgcagg gaactacagt gcggcactgt ggtttccctc ctggggggag 21180

ctcactttgg agaaggaagt ggacagatct gggctgaaga attccagtgt gaggggcacg 21240

agtcccacct ttcactctgc ccagtagcac cccgccctga cgggacatgt agccacagca 21300

gggacgtcgg cgtagtctgc tcaagtgaga cccagggaat gtgttcactt tgttcccatg 21360

ccatgaagag ggtagggtta ggtagtcaca gacatctttt taaagccctg tctccttcca 21420

ggatacacac aaatccgctt ggtgaatggc aagaccccat gtgaaggaag agtggagctc 21480

aacattcttg ggtcctgggg gtccctctgc aactctcact gggacatgga agatgcccat 21540

gttttatgcc agcagcttaa atgtggagtt gccctttcta tcccgggagg agcacctttt 21600

gggaaaggaa gtgagcaggt ctggaggcac atgtttcact gcactgggac tgagaagcac 21660

atgggagatt gttccgtcac tgctctgggc gcatcactct gttcttcagg gcaagtggcc 21720

tctgtaatct gctcaggtaa gagaataagg gcagccagtg atgagccact catgacggtg 21780

ccttaagagt gggtgtacct aggagttccc attgtggctc agtggtaaca aactcgactg 21840

gtatccatga gggtatgggt ttgatccctg gccttgctca atgggttaag gatccagcat 21900

tgctgtgagc tgtggtatag gttgcagact ctgctcaggt cccatgttgc tgtgattgtg 21960

gtgtaggctg actgttgcag cttcaatttg acccctagcc cgggaatttc cataggccac 22020

acgtgcagca ctaaggaagg aaaaaaaaaa aaaaaaaaaa aagagtgggt gtgcctatag 22080

tgaagaacag atgtaaaagg gaagtgaaag ggattccccc attctgaggg attgtgagaa 22140

gtgtgccaga atattaactt catttgactt gttacaggga aagtaaactt gactttcacg 22200

gacctcctag ttacctggtg cttactatat gtcttctcag agtacctgat tcattcccag 22260

cctggttgac ccatccccct atctctatgg ctatgtttat ccagagcaca tctatctaac 22320

actccagctg atcttcctga cacagctgtg gcaaccctgg atcctttaac caactgtgcc 22380

aggctggaga tcaaacctaa gcctctgcag caacccaagc tgctgcagtc agatttttaa 22440

ccccctgtgc cactgtgggt atctccgata ttttgtatct tctgtgactg agtggtttgc 22500

tgtttgcagg gaaccagagt cagacactat ccccgtgcaa ttcatcatcc tcggacccat 22560

caagctctat tatttcagaa gaaaatggtg ttgcctgcat aggtgagaat cagtgaccaa 22620

cctatgaaaa tgatctcaat cctctgaaat gcattttatt catgttttat ttcctctttg 22680

cagggagtgg tcaacttcgc ctggtcgatg gaggtggtcg ttgtgctggg agagtagagg 22740

tctatcatga gggctcctgg ggcaccatct gtgatgacag ctgggacctg aatgatgccc 22800

atgtggtgtg caaacagctg agctgtggat gggccattaa tgccactggt tctgctcatt 22860

ttggggaagg aacagggccc atttggctgg atgagataaa ctgtaatgga aaagaatctc 22920

atatttggca atgccactca catggttggg ggcggcacaa ttgcaggcat aaggaggatg 22980

caggagtcat ctgctcaggt aagttctgca cataacctcg ggttacaatg atttaagaaa 23040

caactaaggt ggggcaaagg gtagtgaggc atatccatca gagcaaattc cctgaaatac 23100

ggactcagag ggaaccattg tgagattgag gttcccagag gtgtggattt aatgaattag 23160

tgttacctca tgtacaaggt agtatactac cagaaagata aaaattcaga agcgagtttg 23220

cagcaaaact catagggaga acttctttta taaataatat ggagctggat attcagtgca 23280

ccacctgatg accactttat taataaataa agagttcctg ttgtggcgca gcggaaatga 23340

atccgacaaa taatcatgag tttgcgggtt tgatccctga cctcactcag tgggttgggg 23400

atctggtgtt gccatgagct gtggtgtagg tcgcagatgc tgcttggatc ctgctttgct 23460

gtggctgtgg tataggcttg tggctacagc tccgatttga ccgctagcct gggaacctcc 23520

atatgctgcg ggggtggccc tcaaaagcaa aataaataaa taagtaaata aataagtagt 23580

ttaaaaagga caagaagaaa tatatttggt attatattct acagagacaa agataatcac 23640

catgcccgat tgatttttca aggcatataa atgagacgtc atgggagcaa aaatggtcat 23700

aatacaatgc ccttgttttg tgtacatggt aagattttag aaagcattgt gaagtaaaaa 23760

agtgtactca gttataatat attggggaaa acagtactat gagaagtaaa aaaatctaca 23820

tgccggaagt tattttttta atgtctcttt tagagtcgca catgcggcat atggaggttc 23880

ccaggctagg ggtcgaatca gagctatagc cactggctta tggcacagcc acaacaacgc 23940

tagatctgag ccacatcagc gacctatact atagctcatg gcaatgccag atccttaacc 24000

tactgagcca agccatgggt caaatccagg tcctcatgga tcctaggcaa attcatttct 24060

gctgagccac gaagggaact cctcagaagt gattttgatg ttactttctt ttcatgacaa 24120

atctggtaaa gtacatacac atagaaactg aagtgtcaga aagggaaata tttcatttta 24180

aggtaatgta tacaaaacag tggttttacc atctgagtat ctcgctaaat tttaactatc 24240

aaggacaatt gccaaaaaaa aaaaaaaaaa aaaagagaga gagagagaac agaatagggt 24300

tatgaagcta aaatcacagt aatttaggga gaaaaaaatc caaagcatgt aattgataaa 24360

aggctctgag cctttgtttg agatttagaa ttcaacttag aaataccggt ggtattttaa 24420

agcagtccat aagtataaaa tccaaggcta aaaagccaga aggtatttgt agaacaaata 24480

tattttaata agctctacca agtcatccag aagctattaa agaattactg gtcactgaca 24540

tagtgtacct gttttcaagg ccattcttac atcagaataa agggagagca ccctctgaat 24600

cttcagaaaa gatgtgaaag tgctaattct ctatttcatc ccagagttca tgtctctgag 24660

actgatcagt gaaaacagca gagagacctg tgcagggcgc ctggaagttt tttacaacgg 24720

agcttggggc agcgttggca ggaatagcat gtctccagcc acagtggggg tggtatgcag 24780

gcagctgggc tgtgcagaca gaggggacat cagccctgca tcttcagaca agacagtgtc 24840

caggcacatg tgggtggaca atgttcagtg tcctaaagga cctgacacac tatggcagtg 24900

cccatcatct ccatggaaga agagactggc cagcccctca gaggagacat ggatcacatg 24960

tgccagtgag tatccattct ttagcgccac tgttatcttc tgatctacct aagcagaagt 25020

gttataacct ttagataatc cctattctac ctggatgatg agattcattc tgtttaattt 25080

ggtgtgcagg tattcagcat cagtgatcat tttcccaaag accatcatgc tctgatggtc 25140

ttctcaaaag ttctaatcag ttgcttcctc cgtgaacagt tgaggagcag agaatatgta 25200

attcagaatt tgactattga atcatcccat ttttctttca tagtcttttg ttgcactgaa 25260

tataaggaga gaagcagtca gaaagatcaa tcctgaatta tttctccatt ctacatctgt 25320

tttaaatttc aaaaaaaaaa ttgttatagg tgatttacaa tgtctgtcaa tttctgctct 25380

acagcaaagt gacccagtta tttgcatata cattcttttt ctcatatttt taaaccggga 25440

gatttctatc cacctggcag tttgagggaa tttaacatta tgcatttatg ttaactttat 25500

tcacctgatg ttttctaagt catactgaga ttcttatgtc caggatggaa tacacctggt 25560

ttgctggaaa gacatgtgct ttcataaaga caaattttgg aaagaatata aaatttaaaa 25620

ggcccatcaa ataaagtttt aagagatttc aaaaaaaagt ttcatctctc tcttttcctc 25680

tttgacctct tgggcgtgtt catcttctca aaaatgatct tggtgtttct gacttttcag 25740

acaaaataag acttcaagaa ggaaacacta attgttctgg acgtgtggag atctggtacg 25800

gaggttcctg gggcactgtg tgtgacgact cctgggacct tgaagatgct caggtggtgt 25860

gccgacagct gggctgtggc tcagctttgg aggcaggaaa agaggccgca tttggccagg 25920

ggactgggcc catatggctc aatgaagtga agtgcaaggg gaatgaaacc tccttgtggg 25980

attgtcctgc cagatcctgg ggccacagtg actgtggaca caaggaggat gctgctgtga 26040

cgtgctcagg tgagggcaga gagtctggat tgagcttgga agctctggca gcaaagagag 26100

ggtgggcggt gacctgcatt gggtaaagat cagaaggtcc agcctaagga tctggtgggg 26160

ggagggacat gatgtttcag tctgaagaat gatgaaaacc tgtgttgtta cgcatgggcc 26220

ttcgccgagg aaagggacat aacttacatg tatcctcctg cagagggagg aagaactagg 26280

ggattctagt tttgtgtggg aaggagcagt ttacttggct caggaggcac taaaggctca 26340

gataggaaac agagatctgt tccattctta ctcccagaac tgattctctt ctcttttctc 26400

ctacagaaat tgcaaagagc cgagaatccc tacatgccac aggtatatca aaaagtttaa 26460

gaacatggga cccattgtct gcattttgtg gaatccctct tattaagaca ttctgggtca 26520

gaagttctga ggatttgaca tttacttcag ctatctgtta tcttacccaa gagagggatg 26580

gtaactagga acccaggtct tttagctaag acattatcac ctcttgtgat gtttacttgt 26640

tctcaggtcg ctcatctttt gttgcacttg caatctttgg ggtcattctg ttggcctgtc 26700

tcatcgcatt cctcatttgg actcagaagc gaagacagag gcagcggctc tcaggtctga 26760

acaaaattac ggtctctcta atgtttctat gggataagaa gcctctctgg ataataaaac 26820

aaaaaaatta cattcaagta tcagttggcc agaaagaggg aacctagaag aggtttaagc 26880

agtttctccg aaacagggaa caagaattca gagaagaaaa ggcacattgg ctgtactgat 26940

gatacctgca ctcgctatgt atgtttaatg ggggacagta gagaattgat agtttagaag 27000

gagtatgctt atatggttct ggatgaatcc tgtatccccc caaacattta ttttctctta 27060

ctatatactt attactaatt taactcttct gtcaagccgt gtgctaggtt ctgaagatgg 27120

ttcagacttg gatactcaag tgcttttgtt ttcatggaat ttccagttta gtggaagaga 27180

taaatatgta aacaaataaa ttgcaatgtt ttattataca ttcgtgtgaa taaggaacaa 27240

aggaggcaca gagaataaag taattactga aaggggaagg ggagtatcag agacttctaa 27300

gtttggaggc agattttgaa gacagaaatc aaagtactgg gtaagatgca tttcaggaaa 27360

gaagaaaaat atgtacacgt gtagagaagc ttaaaagagg gcacacttgt tgttttggag 27420

gggagtacaa gttgagttaa agagagaagt ttctgttaag gctgaagaat agggaagata 27480

cacgtagcga tgctctgtgt tgcatgataa gaagagtcgg agttattaaa gagtatgaga 27540

taggggagtg agataggcag gcaggtcctt agaaagttct gtttggaaat gggatgtcgg 27600

aggggttgaa agagaaccat atattgacaa ggagagcatt ttgaagtagt tgtgatgaaa 27660

gataaaatgg actttatagt gagaatggct gggaaaggat agattttata caaatctcca 27720

atgaattaca gaagaatgct acctgtcttt ggggaagaaa cagggttatc cgatggcatc 27780

ctgttgcgtt tgagttcgtg acatcatgag ggaaaggctt ggcagcgttt actcggtact 27840

gtgtggtaac ttatatggaa aaaaatatga gaaggaatga gtgtgtgtat aactaattta 27900

cttagctgta tgcctgaaat taatacaatt ttataagtca actctactcc aataaaacaa 27960

acaaataaat aaataatttt aactacctga acaaaaaaaa agaatggact ggagacaagt 28020

caaaagtatg gatgatgact acgttatgct tgcactgctg gggaaaagca cacataggga 28080

gggaacgttt tattatgacc cagtccctaa cctatgacct ctgttatcag ttttctcagg 28140

aggagagaat tctgtccatc aaattcaata ccgggagatg aattcttgcc tgaaagcaga 28200

tgaaacggat atgctaaatc cctcaggtcc gtgggttctt tgagggcctg tagccctggg 28260

gttcagatca gcagctgcag ttgaggttga ggcatgctac tttgcacagc agtagaaaga 28320

aatctcaact gtaataggaa gcttgggatg catatgagga agaaaggcaa gaatgaacca 28380

caaattattc ttagggaaga taaaaattgc agtcatgggg agacctctgg ctgagagggc 28440

cgtgattatt tctgacagag ggattatgga gtagaatatg atggcttgga ccttttttca 28500

ctaaaacaag tcagtcttct caaaggtagt ttagcttttc atatatcttt ctcagtttct 28560

tccattccca tttcctgcca ttttcctttc tctaactttt atttattata ttttttccta 28620

aaagtttaaa ttttctatat ctttatccct tcagaagcca tccctagtca caggactagt 28680

tttatttccc attatgtaat gcttctttct ctgtctgttg acttctattt agaaccagtg 28740

cactaaatct gcctctagga acatacctct gctaggttgc aagaaatatc ccattcccca 28800

ctcactctgt gaagactcaa tgcttctcaa tattccttac ctcctgagag ggacttgcct 28860

cacttcttta atccaaggga ctcgattttt gccaaaacta agtcaggaaa acctacataa 28920

gacataggaa agacttgctg tgcttcttaa accccactgt ttgttttcct aattgtgaac 28980

agtattttta aagttcaaag agcttctaag gcacttgagg ggagatctga tttatttccc 29040

agtaattatt ttattccttt cagaaaattc caatgaataa gatggtttta atgatgtggg 29100

actaattttt gtgtctaaat ctcttcctat ttctggatga aaaaaaggag accactctga 29160

agtacaatga aaaggaaaat gggaattata acctggtgag gtgagtaaaa agaatttatt 29220

catcattgct gaaaacaggt acattccttt tgaaagttgg gaactcctct ggtattagaa 29280

aaaaaaaaaa gaacgtatat acacatatat ttccatgtct atgtttatgt ttgtaaatcc 29340

atattcagaa tatgcaacaa ctttttataa ctatgacttc agtccatctt ttagttacat 29400

atatattcta aacaacaact attgctaaga gaagctgggt aagtaaatgt gaataaatct 29460

tctaaagata ttacaggaag ttcctgctgc ggctcagtgg gttaaggact tgatgtcttt 29520

gtgaagatga gggctcgagc cctggcctca ctcagtgagt taaggatcta gcattgctgt 29580

aagctgcagc gtaggttgca gatggggctc agatccagtg ttgctgtggc tgtggcctca 29640

gttgcagctc tgattcaacc cttaggcgag gaacttccat atgcagcaaa tgtggccatt 29700

aaaaaaaaac aaaaaacatt ataggagtca tttcataaaa gagataagac gtttctatag 29760

ttatatagtg catactctgg taaagatagt ataggatact ataggaatat agaaagcttg 29820

cctatgaaaa tttgggaaga ttgtggaaaa gacatctcaa aatatggcat agaaaagaat 29880

catatctttg aggaacagta agtttttcat tcaaaaccgt gtattgaaca tacttatggt 29940

gacaaatggt gtcttgagta ctaaaaattc agtgataaaa gatgctcttg acaaagacat 30000

ggctgttgaa tagaaggtct cactgtcaat gtgtgggaat tatggacagc ctatgtggac 30060

acagggaata gatgagactc taggctggaa ggctgcattg agcccagtaa tgaatggtcc 30120

tgtctgatat atttcatgct catattttat tttagggact attggggagg tggtgggctt 30180

tggaagatta agctgaggca agacacaatc agattgcctt ttataattta ctttcaggag 30240

gaaaatctaa ctaaagaaaa aaagtgaata aggcaagaaa cataagttat acatcaaaaa 30300

gaaaaggtag tggagttcct gttgtggctc agtggttaat gaaccctgct aggaaccatg 30360

aggttgtggg ttcgatccct ggccttgctc agtgggttaa ggatccagcg atgccatgag 30420

ttgtggtgta ggtcgcagac cgtggcttgg gtcccgcatt gctgtggcta tggtgttggc 30480

tggcagctgc agacagctct gattagaccc ctagcctggg aacctccata agccacgagt 30540

gtgaccctag aaaagacaaa aaagaaaaaa gaaagaaaga aaaggtagtg aaatgagtta 30600

gtgggtgtat tttgtttttt aaaaaacaat tttaggagga ctatacattt agaaatgata 30660

tatagcaaaa gtaaggtttt ttgtagaaat attatatatg acatatgaga aatacaaatt 30720

gagacaaagg ataaaagttg gaattcttaa aagtatataa atgtagagtg ttagtatcac 30780

acatatagac acatacacat agatctgtta taaattgata tagtacaaaa atgttaatac 30840

gggtatgcaa attgtatata caataatttg tatacacata tacacattat atataatgca 30900

taatatatat ataagccaat tgttaagaca aagacaatat ctaaaaaata attgggcaaa 30960

gctaagaaaa aatagttcat gcaaaatgaa ttttaaatga ctattagata tttttatttt 31020

tactttattt atttatttat tttattatta ttattattat tttttgtctt tttgcctttt 31080

ctagagccac ttgccatggc atatggaggt tcccaggcta ggggtcaaat cggagctgta 31140

gccaccagcc tatgccagag ccacagcaat gcaggatccg agccacgtct gcgacctaca 31200

ccacagctca cggcaacacc ggatccttaa gccaccgagc aaggctaggg atcgaaccca 31260

caacctcatg gttcctagtc ggattcgtta accactgcgc cacgacggga actccactat 31320

tagatatttt taaaatgctg aagttccggg caattcaaat tatgattaac aatgacatat 31380

tgctttatat aaatcagacc aaaaaaaatt taaaacagca ccaattttat tattggcggg 31440

aataaagaga aaaatgtaat ttcaaagatt gctgttggaa atgaggggtg tggtagcttt 31500

tggagaaagc attctggaga cttctattaa tttttttttt ttaagtgctt caaagatcct 31560

ttgatccaac aattctactc ctaaaaattt cttccataca gataaagcca tttgtctgta 31620

tataacaaat agaagagaat tcctttttgc agccttgtta gtagtgcccc caaactggaa 31680

acaaagtgaa tatcagtcag tggggtagtg gctggaaaaa ttttagtgca cccaaccaac 31740

aaagaaaaac catgcacaaa aattcaataa atatcatctc acttttgtgt tcatgttatt 31800

gaatataatt aaacataatg tttacatcta taaaattatc atatgtatac atgtaaagaa 31860

acattaaaac atttttaaca gactgtaaac ttgaggactg tgaatgactt ttgattgata 31920

atctcaaaca tatggatact attctgatgt aataaataat gattaaattt tttccctaaa 31980

gagtaatcac tactgaatcg ttgcctcaga atcatatgga ggtgctttta aaaaaggcat 32040

ttctgcactg atgttctctg gaatagaagt aattcttatg tacactgaag tttgaaaatc 32100

attgcattta agtgttctgt tcaggaaagt agtgtgcttt ttaatatttg tgagtgaatg 32160

agtaacacaa tacattatat cacattttaa tgtaattcta cacatgtgca tatgaagaga 32220

aaagtaacat ttttttctat ttatgtcttt agttcagcct ttaagatacc ttgatgaaga 32280

cctggactat tgaatgagca agaatctgcc tcttacactg aagattacaa tacagtcctc 32340

tgtctcctgg tattccaaag actgctgctg aatttctaaa gaatagattg gtgaatgtga 32400

ctactcaaag ttgtatgtaa gactttcaag ggcattaaat aaaaaagaat attgctg 32457

<210> 2

<211> 321

<212> DNA

<213> Artificial sequence

<400> 2

gaggaaaaga caaggagctg aggctaacgg gtggtgaaaa caagtgctct ggaagagtgg 60

aggtgaaagt gcaggaggag tggggaactg tgtgtaataa tggctgggac atggatgtgg 120

tctctgttgt ttgtaggcag ctgggatgtc caactgctat caaagccact ggatgggcta 180

attttagtgc aggttctgga cgcatttgga tggatcatgt ttcttgtcga gggaatgagt 240

cagctctctg ggactgcaaa catgatggat ggggaaagca taactgtact caccaacagg 300

atgctggagt aacctgctca g 321

<210> 3

<211> 100

<212> DNA

<213> Artificial sequence

<400> 3

atgtttcttg tcgagggaat gttttagagc tagaaatagc aagttaaaat aaggctagtc 60

cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 100

<210> 4

<211> 100

<212> DNA

<213> Artificial sequence

<400> 4

gatggatcat gtttcttgtc gttttagagc tagaaatagc aagttaaaat aaggctagtc 60

cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 100

<210> 5

<211> 100

<212> DNA

<213> Artificial sequence

<400> 5

gatcatgttt cttgtcgagg gttttagagc tagaaatagc aagttaaaat aaggctagtc 60

cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 100

<210> 6

<211> 4204

<212> DNA

<213> Artificial sequence

<400> 6

ttaatacgac tcactatagg ccaccatgga caagaagtac agcatcggcc tggacatcgg 60

caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt 120

caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct 180

gttcgacagc ggcgaaacag ccgagagaac ccggctgaag agaaccgcca gaagaagata 240

caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa 300

ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa 360

gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta 420

ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg 480

gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg 540

cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta 600

caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct 660

gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga 720

gaagaagaat ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt 780

caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga 840

cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc 900

cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat 960

caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct 1020

gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt 1080

cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt 1140

ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa 1200

gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca 1260

ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt 1320

cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt 1380

gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac 1440

catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat 1500

cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag 1560

cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga 1620

gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct 1680

gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat 1740

cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg 1800

cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa 1860

cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat 1920

cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa 1980

gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga 2040

caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa 2100

cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca 2160

ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc 2220

cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg 2280

ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa 2340

gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg 2400

cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta 2460

cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg 2520

gctgtccgac tacgatgtgg accatatcgt gcctcagagc tttctgaagg acgactccat 2580

cgacaacaag gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc 2640

cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat 2700

tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga 2760

taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc 2820

acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga 2880

agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt 2940

ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt 3000

cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga 3060

ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc 3120

taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct 3180

ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat 3240

cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt 3300

gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcagacc 3360

caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg 3420

cggcttcctg tggcccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg 3480

caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag 3540

cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa 3600

ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag 3660

aatgctggcc tctgccaagc agctgcagaa gggaaacgaa ctggccctgc cctccaaata 3720

tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa 3780

tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca 3840

gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc 3900

cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct 3960

gtttaccctg accagactgg gagcccctag agccttcaag tactttgaca ccaccatcga 4020

ccccaagcag tacagaagca ccaaagaggt gctggacgcc accctgatcc accagagcat 4080

caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgacg gatccggcgg 4140

aggcggaagc gggaaaagaa ccgccgacgg cagcgaattc gagcccaaga agaagaggaa 4200

agtc 4204

<210> 7

<211> 120

<212> DNA

<213> Artificial sequence

<400> 7

ttaatacgac tcactatagg atgtttcttg tcgagggaat gttttagagc tagaaatagc 60

aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 120

<210> 8

<211> 102

<212> RNA

<213> Artificial sequence

<400> 8

gauguuucuu gucgagggaa uguuuuagag cuagaaauag caaguuuaaa auaaggcuag 60

uccguuauca acuugaaaaa guggcaccga gucggugcuu uu 102

Claims (2)

1. A method for preparing CD163 double allelic gene mutation cell, which is to edit CD163 gene of pig in vitro fibroblast genome, so that GTCGAGGGAATGA at position 226 and 238 of the third exon of one chromosome of the double allelic gene CD163 is deleted, GTCGAGGGAAT at position 226 and 236 of the third exon of the other chromosome is deleted, and other nucleotide sequences of the gene are not changed, thus obtaining the CD163 double allelic gene mutation cell;

the nucleotide sequence of the third exon is sequence 2;

the gene editing is carried out by adopting a SpRY-Cas9 system;

the SpRY-Cas9 system includes Cas9 protein and sgRNA;

the target sequence of the sgRNA is 1 st-20 th of the sequence 3;

the cas9 protein is a protein coded by nucleotides from 20 th to 4204 th in the sequence 6;

the SpRY-Cas9 system consists of the Cas9 protein mRNA and the sgRNA;

the mRNA of the cas9 protein is a nucleotide sequence consisting of the 20 th to 4204 th positions in the sequence 6 and is transcribed into RNA;

the nucleotide sequence of the sgRNA is sequence 8;

and the gene editing is to introduce the cas9 protein mRNA and the sgRNA into an in vitro pig fibroblast line to obtain the CD163 double-allele mutant cell.

2. A method for preparing a CD163 double allelic mutant pig, wherein the CD163 double allelic mutant cell prepared by the method of claim 1 is transplanted into a sow through a somatic cell nucleus, and the produced offspring is the CD163 double allelic mutant pig.

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