CN115232816A - CRISPR system for constructing cataract model pig nuclear transplantation donor cells with YAP1 gene mutation and application thereof - Google Patents

CRISPR system for constructing cataract model pig nuclear transplantation donor cells with YAP1 gene mutation and application thereof Download PDF

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CN115232816A
CN115232816A CN202110959122.3A CN202110959122A CN115232816A CN 115232816 A CN115232816 A CN 115232816A CN 202110959122 A CN202110959122 A CN 202110959122A CN 115232816 A CN115232816 A CN 115232816A
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yap1
protein
cataract
leu
lys
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牛冬
汪滔
陶裴裴
刘璐
曾为俊
王磊
程锐
赵泽英
马翔
黄彩云
段星
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Nanjing Qizhen Genetic Engineering Co Ltd
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Abstract

The invention discloses a CRISPR system for constructing cataract model pig nuclear transplantation donor cells with YAP1 gene mutation and application thereof. The present invention provides SEQ ID NO:18, YAP1-gRNA2 shown in SEQ ID NO:19, application of YAP1-gRNA3 and NCN proteins in preparation of a kit; the application of the kit is as follows: preparing a recombinant cell; preparing a cataract model pig; preparing a cataract cell model or a cataract tissue model or a cataract organ model. The invention adopts CRISPR/Cas9 technology combined with double gRNA editing to knock out YAP1 gene, simulates the genetic characteristics of natural onset of cataract, obtains single cell clone of YAP1 gene knock-out, and lays a foundation for cultivating cataract model pigs by somatic cell nuclear transfer animal cloning technology in the later period. The invention has great application value for researching and developing cataract drugs and disclosing pathogenesis of the cataract.

Description

CRISPR system for constructing cataract model pig nuclear transplantation donor cells with YAP1 gene mutation and application thereof
Technical Field
The invention belongs to the technical field of biology, particularly belongs to the technical field of gene editing, and more particularly relates to a CRISPR (clustered regularly interspaced short palindromic repeats) system for constructing cataract model pig nuclear transplantation donor cells with YAP1 gene mutation and application thereof.
Background
Cataract (Cataract) is the most common disease which causes lenticular opacity and finally causes blindness, is an eye disease with the highest blindness causing rate in China, and has the blindness causing rate up to 47 percent. The occurrence of cataract is the result of the combined action of various factors, and the exact pathogenesis of cataract is not clear. To date, the formation of cataracts can be attributed to a combination of factors such as unregulated cell proliferation and apoptosis, abnormal lens fibrocyte differentiation and enucleation, cellular senescence, oxidative stress, and upregulation of inflammatory factors in the eye.
YAP1 is a downstream transcription factor of the Hippo signaling pathway (Hippo pathway) and is involved in development, growth, repair and homeostasis. Early researchers found that YAP1 was in an inactive state when the Hippo signaling pathway was normal; YAP1 is in a hyperactivated state when certain molecules in the Hippo signaling pathway are mutated. At this time, YAP1 in a super-activated state can promote cell proliferation, metastasis, survival, and maintain stem cell activity. It has been shown that YAP1 contributes to lens epithelial development, and that YAP1 is involved in cell environment-dependent transition from a proliferative state to a differentiated state by integrating cell density information in developing lens proteins. Heterozygous inactivation of YAP1 resulted in mouse adult stage cataracts.
So far, no medicine for treating cataract exists in the world, and some medicines only play a role in slowing down the development of cataract but cannot radically reverse the disease. Therefore, the research on the mechanism of occurrence and development of cataract caused by YAP1 gene mutation and the development of corresponding drugs are in need of being carried out on the basis of animal models. The current common animal model is a mouse model, however, the mouse is different from the human body in the aspects of body type, organ size, physiology, pathology and the like, and the normal physiological and pathological states of the human body cannot be simulated really. The pig as a large animal has similar body size and physiological function to human, is easy to breed and feed in large scale, has lower requirements on ethics, animal protection and the like, and is an ideal human disease model animal.
Gene editing is a biotechnology that has been greatly developed in recent years, and includes editing technologies from homologous recombination-based gene editing to nuclease-based ZFNs, TALENs, CRISPR/Cas9, and the like, wherein CRISPR/Cas9 technology is currently the most advanced gene editing technology. Currently, gene editing techniques are increasingly applied to the production of animal models.
Disclosure of Invention
The invention aims to provide a CRISPR system for constructing cataract model pig nuclear transplantation donor cells with YAP1 gene mutation and application thereof.
The invention provides application of YAP1-gRNA2, YAP1-gRNA3 and NCN proteins in preparation of a kit.
The invention also provides application of YAP1-gRNA2, YAP1-gRNA3 and PRONCN proteins in preparation of the kit.
The invention also provides application of YAP1-gRNA2, YAP1-gRNA3 and the ultra-heterogeneous particles in preparation of the kit.
The invention also provides a method for preparing recombinant cells, which comprises the following steps: and co-transfecting the YAP1-gRNA2, the YAP1-gRNA3 and the NCN protein to the pig cells to obtain recombinant cells.
The invention provides a kit, which comprises YAP1-gRNA2, YAP1-gRNA3 and NCN protein.
The invention provides a kit, which comprises YAP1-gRNA2, YAP1-gRNA3 and PRONCN protein.
The invention provides a kit which comprises YAP1-gRNA2, YAP1-gRNA3 and specific plasmids.
Any one of the kits above further comprising porcine cells.
The use of any one of the above kits is (a) or (b) or (c): (a) preparing a recombinant cell; (b) preparing cataract model pig; (c) Preparing a cataract cell model or a cataract tissue model or a cataract organ model.
The co-transfection is specifically a shock transfection.
The parameter settings of the electroporation transfection can be specifically as follows: 1450V, 10ms, 3pulse.
The co-transfection may be specifically carried out using a mammalian nuclear transfection kit (Neon kit, thermofoisher) and a Neon TM transfection system electrotransfer apparatus.
The ratios of YAP1-gRNA2, YAP1-gRNA3 and NCN protein are as follows in sequence: 0.8-1.2 μ g YAP1-gRNA2:0.8-1.2 μ g YAP1-gRNA3: 3-5. Mu.g NCN protein.
The ratios of YAP1-gRNA2, YAP1-gRNA3 and NCN protein are as follows in sequence: 1 μ g YAP1-gRNA2:1 μ g YAP1-gRNA3: mu.g NCN protein.
The proportions of the pig cells, YAP1-gRNA2, YAP1-gRNA3 and NCN protein are as follows: 10 ten thousand porcine cells: 0.8-1.2 μ g YAP1-gRNA2:0.8-1.2 μ g YAP1-gRNA3: 3-5. Mu.g NCN protein.
The proportions of the pig cells, YAP1-gRNA2, YAP1-gRNA3 and NCN protein are as follows: 10 ten thousand porcine cells: 1 μ g YAP1-gRNA2:1 μ g YAP1-gRNA3: mu.g NCN protein.
Any one of the YAP1-gRNA2 is sgRNA, and a target sequence binding region thereof is as set forth in SEQ ID NO:18 from nucleotide 3 to nucleotide 22.
Specifically, the YAP1-gRNA2 is shown as SEQ ID NO:18, respectively.
Specifically, the YAP1-gRNA2 is shown as SEQ ID NO: shown at 11.
Any one of the YAP1-gRNA3 is sgRNA, and a target sequence binding region thereof is as set forth in SEQ ID NO:19 at nucleotides 3-22.
Specifically, the YAP1-gRNA3 is shown as SEQ ID NO:19, respectively.
Specifically, the YAP1-gRNA3 is shown as SEQ ID NO: shown at 12.
Any of the NCN proteins described above is a Cas9 protein or a fusion protein with a Cas9 protein.
Specifically, the NCN protein is shown as SEQ ID NO:3, respectively.
Any one of the above porcine cells is a porcine fibroblast.
Any one of the above porcine cells is a porcine primary fibroblast.
The preparation method of the NCN protein comprises the following steps:
(1) Introducing the plasmid pKG-GE4 into escherichia coli BL21 (DE 3) to obtain a recombinant strain;
(2) Culturing the recombinant bacteria by adopting a liquid culture medium at 30 ℃, then adding IPTG (isopropyl-beta-D-thiogalactoside) and carrying out induced culture at 25 ℃, and then collecting bacteria;
(3) Crushing the collected thalli, and collecting a crude protein solution;
(4) Purification of the crude protein solution with His by affinity chromatography 6 A tagged fusion protein;
(5) By using a compound having His 6 Tagged enterokinase cleavage with His 6 The tagged fusion protein was then removed with His using Ni-NTA resin 6 A tagged protein, resulting in a purified NCN protein;
plasmid pKG-GE4 has the sequence shown in SEQ ID NO:1, 5209 to 9852 th nucleotide.
The preparation method of the NCN protein specifically comprises the following steps:
(1) The plasmid pKG-GE4 was introduced into E.coli BL21 (DE 3) to obtain a recombinant strain.
(2) Inoculating the recombinant bacteria obtained in the step (1) to a liquid LB culture medium containing ampicillin, and carrying out shake culture;
(3) Inoculating the bacterial liquid obtained in the step (2) to a liquid LB culture medium, and carrying out shaking culture at 30 ℃ and 230rpm until the bacterial liquid is OD 600nm The value =1.0, then IPTG was added to make the concentration in the system 0.5mM, followed by shaking culture at 230rpm at 25 ℃ for 12 hours, and then the cells were collected by centrifugation;
(4) Taking the thalli obtained in the step (3), and washing the thalli with a PBS (phosphate buffer solution);
(5) Adding the crude extraction buffer solution into the thalli obtained in the step (4), suspending the thalli, then crushing the thalli, then centrifugally collecting supernate, filtering by adopting a filter membrane with the aperture of 0.22 mu m, and collecting filtrate;
(6) Purifying the filtrate obtained in step (5) by affinity chromatography to obtain a purified product having His 6 A fusion protein of the tag (the fusion protein shown in SEQ ID NO: 2);
(7) Taking the solution after column chromatography collected in the step (6), concentrating by using an ultrafiltration tube, and then diluting with 25mM Tris-HCl (pH8.0);
(8) Will have His 6 Adding the labeled recombinant bovine enterokinase into the solution obtained in the step (7), and performing enzyme digestion;
(9) Mixing the solution obtained in the step (8) with Ni-NTA resin, incubating, and centrifuging to collect supernatant;
(10) And (4) concentrating the supernatant obtained in the step (9) by using an ultrafiltration tube, and then adding the concentrated supernatant into an enzyme stock solution to obtain the NCN protein solution.
Purifying the filtrate obtained in step (5) to give a purified product having His by affinity chromatography 6 The specific method of the tagged fusion protein is as follows:
firstly, balancing a Ni-NTA agarose column by using a balance solution with 5 column volumes (the flow rate is 1 ml/min); then, 50ml of the filtrate obtained in the step (5) is loaded (the flow rate is 0.5-1 ml/min); the column was then washed with 5 column volumes of equilibration solution (flow rate 1 ml/min); the column was then washed with 5 column volumes of buffer (flow rate 1 ml/min) to remove contaminating proteins; then eluting with 10 column volumes of eluent at flow rate of 0.5-1ml/min, and collecting the solution (90-100 ml) after passing through the column.
Any one of the PRONCN proteins sequentially comprises the following elements from upstream to downstream: signal peptide, molecular chaperone protein, protein tag, protease cleavage site, nuclear localization signal, cas9 protein, nuclear localization signal.
The signal peptide has the function of promoting protein secretion expression. The signal peptide may be selected from the group consisting of the escherichia coli alkaline phosphatase (phoA) signal peptide, the staphylococcus aureus protein a signal peptide, the escherichia coli outer membrane protein (ompa) signal peptide or the signal peptide of any other prokaryotic gene, preferably the alkaline phosphatase signal peptide (phoA signal peptide). The signal peptide of alkaline phosphatase is used to guide the secretory expression of the target protein into the bacterial periplasm cavity so as to be separated from the protein in the bacterial cell, and the target protein secreted into the bacterial periplasm cavity is soluble expression and can be cleaved by the signal peptidase in the bacterial periplasm cavity.
The chaperone protein functions to increase the solubility of the protein. The chaperone may be any protein that assists in the formation of disulfide bonds, preferably a thioredoxin (TrxA protein). The thioredoxin can be used as a molecular chaperone to help a co-expressed target protein (such as a Cas9 protein) to form a disulfide bond, so that the stability and the folding correctness of the protein are improved, and the solubility and the activity of the target protein are increased.
The protein tag functions for protein purification. The Tag can be His Tag (His-Tag, his) 6 Protein tag), GST tag, flag tag, HA tag, c-Myc tag, or any other protein tag, more preferably His tag. The His tag can be combined with a Ni column, and the target protein can be purified by one-step Ni column affinity chromatography, so that the purification process of the target protein can be greatly simplified.
The protease cleavage site functions to cleave non-functional segments after purification to release the native form of the Cas9 protein. The protease may be selected from Enterokinase (Enterokinase), factor Xa (Factor Xa), thrombin (thrombobin), TEV protease (TEV protease), HRV 3C protease (HRV 3C protease), WELQut protease or any other endoprotease, further preferably Enterokinase. EK is an enterokinase enzyme cutting site, so that fused TrxA-His segment can be conveniently cut by enterokinase to obtain the Cas9 protein in a natural form. After the fusion protein is digested by using the commercial enterokinase with the His label, the TrxA-His section and the enterokinase with the His label can be removed through one-time affinity chromatography to obtain the Cas9 protein in a natural form, so that the damage and loss of target protein caused by multiple times of purification and dialysis are avoided.
The nuclear localization signal may be any nuclear localization signal, preferably an SV40 nuclear localization signal and/or a nucleocapsin nuclear localization signal. NLS is a nuclear localization signal, and NLS sites are respectively designed at the N end and the C end of Cas9, so that Cas9 can more effectively enter a cell nucleus for gene editing.
The Cas9 protein may be saCas9 or spCas9, preferably a spCas9 protein.
The PRONCN protein is specifically shown as SEQ ID NO:2, respectively.
Any one of the specific plasmids comprises the following elements from upstream to downstream in sequence: promoter, operator, ribosome binding site, PRONCN protein coding gene and terminator.
The promoter may specifically be a T7 promoter. The T7 promoter is a prokaryotic expression strong promoter and can efficiently drive the expression of exogenous genes.
The operon may specifically be a Lac operon. The Lac operon is a regulatory element for lactose induced expression, and after bacteria grow to a certain amount, IPTG is used for inducing the expression of the target protein at low temperature, so that the influence of the premature expression of the target protein on the growth of host bacteria can be avoided, and the solubility of the expressed target protein is also obviously improved by the induced expression at low temperature.
The ribosome binding site is a ribosome binding site for translation of a protein, and is essential for translation of a protein.
The terminator may specifically be a T7 terminator. The T7 terminator can effectively terminate gene transcription at the end of the target gene, and prevent other downstream sequences except the target gene from being transcribed and translated.
For the codon of the spCas9 protein, the codon is optimized, so that the codon preference of the escherichia coli high-efficiency expression strain E.coli BL21 (DE 3) selected by the application is completely adapted, and the expression level of the Cas9 protein is improved.
The T7 promoter is shown as SEQ ID NO:1 from nucleotide 5121 to nucleotide 5139.
The Lac operon is shown as SEQ ID NO:1 from nucleotide 5140 to nucleotide 5164.
The ribosome binding site is shown as SEQ ID NO:1 from nucleotide 5178 to 5201.
The coding sequence of the alkaline phosphatase signal peptide is shown as SEQ ID NO:1, nucleotides 5209-5271.
The coding sequence of the TrxA protein is shown as SEQ ID NO:1, nucleotides 5272-5598.
The coding sequence of His-Tag is shown as SEQ ID NO:1, nucleotides 5620-5637.
The coding sequence of the enterokinase enzyme cutting site is shown as SEQ ID NO:1 from nucleotide 5638 to 5652.
The coding sequence of the nuclear localization signal is shown as SEQ ID NO:1 from nucleotide 5656 to nucleotide 5670.
The coding sequence of the spCas9 protein is shown in SEQ ID NO:1, nucleotides 5701-9801.
The coding sequence of the nuclear localization signal is shown as SEQ ID NO:1, nucleotides 9802 to 9849.
The T7 terminator is shown as SEQ ID NO:1, nucleotides 9902-9949.
Specifically, the specific plasmid is plasmid pKG-GE4.
Plasmid pKG-GE4 has the sequence SEQ ID NO:1, nucleotides 5121-9949.
Specifically, any one of the plasmids pKG-GE4 is shown as SEQ ID NO:1 is shown.
The invention also protects the recombinant cell prepared by any one of the methods.
The invention also protects the application of the recombinant cell in preparing cataract model pigs.
The recombinant cell is taken as a nuclear transplantation donor cell to carry out somatic cell cloning, so that a cloned pig, namely the cataract model pig can be obtained.
The invention also protects the pig tissue of the model pig prepared by the recombinant cell, namely a cataract tissue model.
The invention also protects a pig organ of a model pig prepared by the recombinant cell, namely a cataract organ model.
The invention also protects the pig cell of the model pig prepared by the recombinant cell, namely a cataract cell model.
The invention also protects the application of the recombinant cell, the cataract tissue model, the cataract organ model, the cataract cell model or the cataract model pig, which is (d 1) or (d 2) or (d 3) or (d 4) as follows:
(d1) Screening the medicine for treating cataract;
(d2) Evaluating the drug effect of the cataract drug;
(d3) Evaluating the curative effect of gene therapy and/or cell therapy of cataract;
(d4) The pathogenesis of cataract is studied.
Any one of the pigs may be a Yuanjiang fragrant pig.
Any of the above cataracts are caused by mutations in the YAP1 gene.
Pig YAP1 gene information: encodes YES 1-related transcription factor 1; is located on chromosome 9; geneID is 100622418, sus scrofa.
The amino acid sequence coded by the pig YAP1 gene is shown as SEQ ID NO: shown in fig. 8.
The pig YAP1 gene has the nucleotide sequence shown in SEQ ID NO:9, or a fragment of DNA as set forth in seq id no.
Any one of the recombinant cells is a recombinant cell with YAP1 gene mutation.
Any of the above mutations is a deletion and/or insertion and/or substitution of one or more nucleotides.
Any one of the above recombinant cells is a single cell clone of a heterozygous type.
Any of the above recombinant cells may specifically be a single cell clone whose genotype is heterozygous in table 1.
Compared with the prior art, the invention has at least the following beneficial effects:
(1) The subject of the invention (pig) has better applicability than other animals (rats, mice, primates).
Rodents such as rats and mice have great differences from humans in body types, organ sizes, physiology, pathology and the like, and cannot truly simulate normal physiological and pathological states of humans. Studies have shown that over 95% of drugs validated to be effective in large mice are not effective in human clinical trials. In large animals, primates are animals that have a close relationship with humans, but are small in size, late in sexual maturity (mating starts at age 6-7), and are single-birth animals, and the population propagation speed is extremely slow, and the raising cost is high. In addition, primate cloning efficiency is low, difficulty is high, and cost is high.
However, pigs, which are animals related to humans other than primates, do not have the above-mentioned disadvantages, and have body types, body weights, organ sizes, and the like similar to those of humans, and are very similar to those of humans in terms of anatomy, physiology, immunology, nutritional metabolism, disease pathogenesis, and the like. Meanwhile, the pigs have early sexual maturity (4-6 months), high reproductive capacity and multiple piglets in one birth, and can form a large group within 2-3 years. In addition, the cloning technology of the pig is very mature, and the cloning and feeding cost is much lower than that of a primate. Pigs are therefore very suitable animals as models for human diseases.
(2) The vector constructed by the invention uses a strong promoter T7-lac which can express target protein with high efficiency to express the target protein, and uses a signal peptide of bacterial periplasmic protein alkaline phosphatase (phoA) to guide the secretion and expression of the target protein to a bacterial periplasm cavity, so that the target protein is separated from the bacterial intracellular protein and is expressed in a soluble way. Meanwhile, the thioredoxin TrxA and the Cas9 protein are fused and expressed, the TrxA can help the coexpressed target protein to form a disulfide bond, the stability and the folding correctness of the protein are improved, and the solubility and the activity of the target protein are increased. In order to facilitate the purification of the target protein, the His label is designed, and the target protein can be purified through one-step Ni column affinity chromatography, so that the purification process of the target protein is greatly simplified. Meanwhile, an enterokinase enzyme cutting site is designed behind the His tag, so that the fused TrxA-His polypeptide fragment can be conveniently cut off, and the Cas9 protein in a natural form can be obtained. After the fusion protein is digested by using the enterokinase with the His tag, the TrxA-His polypeptide fragment and the enterokinase with the His tag can be removed through one-time affinity chromatography to obtain the Cas9 protein in a natural form, so that the damage and loss of the target protein caused by multiple times of purification dialysis are avoided. Meanwhile, the invention also designs an NLS site at the N end and the C end of the Cas9 respectively, so that the Cas9 can enter the cell nucleus more effectively for gene editing. In addition, the E.coli BL21 (DE 3) strain is selected as a target protein expression strain, and the strain can efficiently express and clone a foreign gene of an expression vector (such as pET-32 a) containing a bacteriophage T7 promoter. Meanwhile, as for the codon of the Cas9 protein, the codon optimization is carried out, so that the codon preference of the expression strain is completely adapted, and the expression level of the target protein is improved. In addition, after the bacteria grow to a certain amount, IPTG is used for inducing the expression of the target protein at low temperature, so that the influence of the premature expression of the target protein on the growth of host bacteria can be avoided, and the solubility of the expressed target protein is also obviously improved by inducing the expression at low temperature. Through the optimization design and experimental implementation, the activity of the obtained Cas9 protein is remarkably improved compared with that of a commercial Cas9 protein.
(3) The gene editing is carried out by combining the Cas9 high-efficiency protein constructed and expressed by the invention with the gRNA transcribed in vitro, and the optimal dosage ratio of the Cas9 and the gRNA is optimized, so that the ratio of the obtained single cell clone for gene editing is up to 83.7 percent and is far higher than the conventional gene editing efficiency (10-30 percent).
(4) The cloned pig with the knocked-out target gene can be directly obtained by cloning somatic cell nuclear transfer animals by using the obtained single cell cloned strain with the knocked-out target gene, and the gene variation can be stably inherited.
The method for embryo transplantation after injecting gene editing materials into germ cells in micro-injection in mouse model production is not suitable for producing large animal (such as pig) models with longer gestation period because the probability of directly obtaining gene mutation offspring is lower and the offspring hybridization breeding is needed. Therefore, the method adopts the primary cell in-vitro editing with great technical difficulty and high challenge, the double gRNA cutting and the screening of the positive editing single cell clone, and the corresponding disease model pig is directly obtained through the somatic cell nuclear transfer animal cloning technology in the later stage, so that the model pig manufacturing period can be greatly shortened, and the manpower, material resources and financial resources are saved.
The invention adopts CRISPR/Cas9 technology combined with double gRNA editing to knock out YAP1 gene, simulates the genetic characteristics of natural onset of cataract, obtains single cell clone of YAP1 gene knock-out, and lays a foundation for cultivating cataract model pigs by somatic cell nuclear transfer animal cloning technology in later period. The invention is helpful for researching and disclosing the pathogenesis of the cataract caused by YAP1 gene dysfunction, can also be used for researching drug screening, drug effect evaluation, gene therapy, cell therapy and the like, can provide effective experimental data for further clinical application, and further provides powerful experimental means for successfully treating the human cataract. The invention has great application value for researching and developing cataract drugs and disclosing pathogenesis of the cataract.
Drawings
FIG. 1 is a schematic diagram of the structure of plasmid pET-32 a.
FIG. 2 is a schematic diagram of the structure of plasmid pKG-GE4.
FIG. 3 is an alignment of reverse sequencing of single cell clone numbered 4 with the wild type sequence.
FIG. 4 is an alignment of reverse sequencing of single cell clone numbered 2 to the wild type sequence.
FIG. 5 shows the forward and reverse sequencing of single cell clone numbered 1 aligned simultaneously with the wild type sequence.
FIG. 6 is an alignment of reverse sequencing of single cell clone number 6 with the wild type sequence.
FIG. 7 is an electrophoretogram of PCR amplification using different primer pairs using ear tissue-extracted genomes of swine designated as 1 as templates in example 4.
FIG. 8 is an electrophoretogram of PCR amplification in example 4 using genomic DNA of 18 pigs as templates and primer pairs consisting of YAP1-E2g-JDF132 and YAP1-E2g-JDR570, respectively.
FIG. 9 is an electropherogram comparing the editing efficiency of different target combinations in example 4.
FIG. 10 is an electrophoretogram of example 5 in which the ratio of gRNA to NCN protein is optimized.
Fig. 11 is an electrophoretogram comparing gene editing efficiency of NCN protein and a commercial Cas9 protein in example 5.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The recombinant plasmids constructed in the examples were all sequence verified. The commercial Cas9-a protein is a commercially available Cas9 protein with good effect. The commercial Cas9-B protein is a commercial Cas9 protein with good effect. Complete culture broth (% by volume): 15% fetal bovine serum (Gibco) +83% DMEM medium (Gibco) +1% Penicilin-Streptomyces (Gibco) +1% HEPES (Solarbio). Cell culture conditions: 37 ℃ C., 5% CO 2 、5％O 2 The constant temperature incubator.
Plasmid pKG-GE3, a circular plasmid, as described in patent application 202010084343.6, SEQ ID NO:2, respectively. SEQ ID NO:2, the nucleotide at positions 395 to 680 constitutes CMV enhancer, the nucleotide at positions 682 to 890 constitutes EF1a promoter, the nucleotide at positions 986 to 1006 encodes a Nuclear Localization Signal (NLS), the nucleotide at positions 1016 to 1036 encodes a Nuclear Localization Signal (NLS), the nucleotide at positions 1037 to 5161 encodes Cas9 protein, the nucleotide at positions 5162 to 5209 encodes a Nuclear Localization Signal (NLS), the nucleotide at positions 5219 to 5266 encodes a Nuclear Localization Signal (NLS), the nucleotide at positions 5276 to 5332 encodes self-cleaving polypeptide P2A (the amino acid sequence of self-cleaving polypeptide P2A is "ATNFSLSLLKKQAKGDAKGDVEENPGP", the position of self-cleaving is between the first and second amino acid residues from the C-terminus of the sequence), the nucleotide at positions 5333 to 6046 encodes EGFP protein, the nucleotide at positions 6056 to 539 encodes self-cleaving polypeptide T2A (the amino acid sequence of self-cleaving polypeptide T2A is "EGSLRGSLRGPLGVEGDVEGFENP", the nucleotide at positions 73610739 and the nucleotide at positions 7373769 to 677647), the nucleotide at positions WPBYb 6747 encodes the nucleotide sequence (the nucleotide sequence of the nucleotide at positions WPSbSLRGBW 679) and the nucleotide sequence of the nucleotide at positions 677610 to 677647), and the nucleotide sequence of WPSbRGSLRG 677610 to 677647, the sequence (WPSbRG 679). SEQ ID NO:2, the 911-6706 th nucleotides form fusion gene to express fusion protein. Due to the presence of the self-cleaving polypeptide P2A and the self-cleaving polypeptide T2A, the fusion protein spontaneously forms the following three proteins: proteins with Cas9 protein, proteins with EGFP protein and proteins with Puro protein.
The pKG-U6gRNA vector, plasmid pKG-U6gRNA, is a circular plasmid, as described in patent application 202010084343.6, SEQ ID NO:3, respectively. SEQ ID NO:3, the 2280 th to 2539 th nucleotides form the hU6 promoter, and the 2558 th to 2637 th nucleotides are used for transcription to form a gRNA framework. When the recombinant plasmid is used, a DNA molecule (a target sequence binding region for forming gRNA through transcription) of about 20bp is inserted into the plasmid pKG-U6gRNA to form a recombinant plasmid, and the recombinant plasmid is transcribed in a cell to obtain the gRNA.
The porcine primary fibroblasts used in the examples were all prepared from porcine ear tissue, which was freshly obtained from Jiangxiang pigs. The method for preparing the primary pig fibroblasts comprises the following steps: (1) taking 0.5g of pig ear tissue, removing hair and bone tissue, soaking in 75% alcohol for 30-40s, washing with PBS buffer containing 5% (volume ratio) Penicillin-Streptomycin (Gibco) for 5 times, and washing with PBS buffer for one time; (2) shearing the tissue with scissors, digesting with 5mL of 0.1% collagenase solution (Sigma) at 37 ℃ for 1h, centrifuging at 500g for 5min, and discarding the supernatant; (3) resuspending the precipitate with 1mL of complete culture solution, spreading into a 10cm diameter cell culture dish containing 10mL of complete culture solution and sealed with 0.2% gelatin (VWR), and culturing until the bottom of the dish is 60% full of cells; (4) after completion of step (3), the cells were digested with trypsin and collected, and then resuspended in complete medium. Used for carrying out subsequent electrotransfer experiments.
Example 1 construction of prokaryotic Cas9 high-efficiency expression vector
The structure of plasmid pET-32a is schematically shown in FIG. 1.
The plasmid pKG-GE4 is obtained by modifying plasmid pET-32a serving as a starting plasmid. Plasmid pET32a-T7lac-phoA SP-TrxA-His-EK-NLS-spCas9-NLS-T7ter (plasmid pKG-GE4 for short), as shown in SEQ ID NO:1, which is circular plasmid, and the structural schematic diagram is shown in figure 2.
The amino acid sequence of SEQ ID NO: in 1, the nucleotide 5121-5139 constitutes T7 promoter, the nucleotide 5140-5164 constitutes Lac operator (Lac operator), the nucleotide 5178-5201 constitutes Ribosome Binding Site (RBS), the nucleotide 5209-5271 constitutes alkaline phosphatase signal peptide (phoA signal peptide), the nucleotide 5272-5598 constitutes TrxA protein, and the nucleotide 5620-5637 constitutes His-Tag (also named His-Tag) 6 Tag), 5638-5652 nucleotides encode enterokinase cleavage site (EK cleavage site), 5656-5670 nucleotides encode nuclear localization signal, 5701-9801 nucleotides encode spCas9 protein, 9802-9849 nucleotides encode nuclear localization signal, and 9902-9949 nucleotides constitute T7 terminator. The nucleotides encoding the spCas9 protein have been codon optimized for the e.coli BL21 (DE 3) strain.
The main modifications of plasmid pKG-GE4 are as follows: (1) the coding region of the TrxA protein is reserved, and the TrxA protein can help the expressed target protein to form a disulfide bond and increase the solubility and the activity of the target protein; adding a coding sequence of an alkaline phosphatase signal peptide in front of a coding region of the TrxA protein, wherein the alkaline phosphatase signal peptide can guide the expressed target protein to be secreted into the periplasmic cavity of the membrane of the bacterium and can be cut by a prokaryotic periplasmic signal peptidase; (2) adding a coding sequence of His-Tag behind the coding sequence of the TrxA protein, wherein the His-Tag can be used for enriching the expressed target protein; (3) adding the coding sequence of an enterokinase enzyme cutting site DDDDK (Asp-Asp-Asp-Asp-Lys) at the downstream of the coding sequence of the His-Tag, and removing the His-Tag and the upstream fused TrxA protein by the purified protein under the action of enterokinase; (4) the Cas9 gene which is suitable for being expressed by an escherichia coli BL21 (DE 3) strain after codon optimization is inserted, and meanwhile, the nuclear localization signal coding sequence is added at the upstream and the downstream of the gene, so that the nuclear localization capability of the Cas9 protein purified at the later stage is improved.
The fusion gene in the plasmid pKG-GE4 is shown as SEQ ID NO:1, nucleotides 5209 to 9852 of SEQ ID NO:2 (fusion protein TrxA-His-EK-NLS-spCas9-NLS, abbreviated as PRONCN protein). Due to the existence of the alkaline phosphatase signal peptide and the enterokinase enzyme cutting site, the fusion protein is cut by enterokinase enzyme to form SEQ ID NO:3, the protein shown in SEQ ID NO:3 is designated as NCN protein.
Example 2 preparation and purification of NCN protein
1. Inducible expression
1. The plasmid pKG-GE4 was introduced into E.coli BL21 (DE 3) to obtain a recombinant strain.
2. The recombinant strain obtained in step 1 was inoculated into a liquid LB medium containing 100. Mu.g/ml ampicillin and cultured overnight at 37 ℃ with shaking at 200 rpm.
3. Inoculating the bacterial liquid obtained in the step 2 to a liquid LB culture medium, and carrying out shaking culture at 30 ℃ and 230rpm until the bacterial liquid is OD 600nm The value =1.0, isopropyl thiogalactoside (IPTG) was added to make the concentration in the system 0.5mM, and the mixture was subjected to shaking culture at 230rpm at 25 ℃ for 12 hours, then centrifuged at 10000 ℃ for 15 minutes at 4 ℃ to collect the cells.
4. The cells obtained in step 3 were washed with PBS buffer.
2. Purification of fusion protein TrxA-His-EK-NLS-spCas9-NLS
1. And (3) adding the crude extraction buffer solution into the thallus obtained in the step one, suspending the thallus, then crushing the thallus by using a homogenizer (1000 par circulation is carried out for three times), then centrifuging for 30min at 4 ℃ at 15000g, collecting supernatant, filtering the supernatant by using a filter membrane with the aperture of 0.22 mu m, and collecting filtrate. In this step, 10ml of crude extraction buffer solution is prepared for each g of wet-weight thallus.
Crude extraction buffer: containing 20mM Tris-HCl (pH 8.0), 0.5M NaCl, 5mM Imidazole, 1mM PMSF, and the balance ddH 2 O。
2. The fusion protein was purified by affinity chromatography.
Firstly, balancing a Ni-NTA agarose column by using a balancing solution with 5 column volumes (the flow rate is 1 ml/min); then 50ml of the filtrate obtained in step 1 was loaded (flow rate 0.5-1 ml/min); the column was then washed with 5 column volumes of equilibration solution (flow rate 1 ml/min); the column was then washed with 5 column volumes of buffer (flow rate 1 ml/min) to remove contaminating proteins; then eluting with 10 column volumes of eluent at a flow rate of 0.5-1ml/min, and collecting the solution (90-100 ml) after passing through the column.
Ni-NTA agarose column: ausrey, L00250/L00250-C, 10ml of filler.
Balance liquid: containing 20mM Tris-HCl (pH 8.0), 0.5M NaCl, 5mM Imidazole, and the balance ddH 2 O。
Buffer solution: containing 20mM Tris-HCl (pH 8.0), 0.5M NaCl, 50mM Imidazole, and the balance ddH 2 O。
Eluent: containing 20mM Tris-HCl (pH 8.0), 0.5M NaCl, 500mM Imidazole, and the balance ddH 2 O。
3. Enzyme digestion of fusion protein TrxA-His-EK-NLS-spCas9-NLS and purification of NCN protein
1. 15ml of the post-column solution collected in step two was concentrated to 200. Mu.l using Amicon ultrafiltration tube (Sigma, UFC9100, 15ml capacity) and then diluted to 1ml with 25mM Tris-HCl (pH 8.0). 6 ultrafiltration tubes were used to give a total of 6ml.
2. The product is obtained from commercial sources and has His 6 Tagged recombinant bovine enterokinase (biol., C620031, recombinant bovine enterokinase light chain, his-bearing 6 The tag, recombinan Bovine Enterokinase Light Chain, his), was added to the solution (about 6 ml) obtained in step 1, and cleaved at 25 ℃ for 16 hours. 2 units of enterokinase are added in the amount of each 50 mug protein.
3. The solution (about 6 ml) that completed step 2 was mixed with 480. Mu.l Ni-NTA resin (Kinseri, L00250/L00250-C), mixed by rotation at room temperature for 15min, then centrifuged at 7000g for 3min, and the supernatant (4-5.5 ml) was collected.
4. And (3) taking the supernatant obtained in the step (3), concentrating the supernatant to 200 mu l by using an Amicon ultrafiltration tube (Sigma, UFC9100, the volume of which is 15 ml), adding the concentrated solution into an enzyme stock solution, and adjusting the protein concentration to be 5mg/ml to obtain the NCN protein solution.
And (3) sequencing the protein in the NCN protein solution, wherein the 15N-terminal amino acid residues are shown as SEQ ID NO:3, positions 1 to 15, i.e., the NCN protein.
The NCN proteins used in the subsequent examples were all provided by NCN protein solutions.
Enzyme stock solution (ph 7.4): containing 10mM Tris,300mM NaCl,0.1mM EDTA,1mM DTT,50% (by volume) glycerol, and the balance ddH 2 O。
Example 3 preparation of YAP1 Gene knockout clone from Jiangxiang pig Single cell
Two high-efficiency gRNA targets (YAP 1-E2-gRNA2 and YAP1-E2-gRNA 3) screened in example 4 were selected.
1. Preparation of gRNA
1. Preparation of YAP1-T7-gRNA2 transcription template and YAP1-T7-gRNA3 transcription template
The YAP1-T7-gRNA2 transcription template is a double-stranded DNA molecule, and is shown as SEQ ID NO: shown at 16.
The YAP1-T7-gRNA3 transcription template is a double-stranded DNA molecule, and is shown as SEQ ID NO: shown at 17.
2. In vitro transcription to obtain gRNA
Taking YAP1-T7-gRNA2 Transcription template, adopting Transcript Aid T7 High Yield Transcription Kit (Fermentas, K0441) to make in vitro Transcription, then using MEGA clear TM The transformation Clean-Up Kit (Thermo, AM 1908) was recovered and purified to obtain YAP1-gRNA2.YAP1-gRNA2 is single-stranded RNA, and is shown in SEQ ID NO:18, respectively.
YAP1-gRNA2(SEQ ID NO：18)：
GGUGAUGAUGUACCUCUGCCAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU
Taking YAP1-T7-gRNA3 Transcription template, adopting Transcript Aid T7 High Yield Transcription Kit (Fermentas, K0441) to make in vitro Transcription, then using MEGA clear TM TranThe description Clean-Up Kit (Thermo, AM 1908) was recovered and purified to obtain YAP1-gRNA3.YAP1-gRNA3 is single-stranded RNA, and is shown in SEQ ID NO: as shown at 19.
YAP1-gRNA3(SEQ ID NO：19)：
GGAGAUAUGGCUCCCAGCUGCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU
2. Transfection of porcine primary fibroblasts
1.YAP1-gRNA2, YAP1-gRNA3 and NCN protein were co-transfected into porcine primary fibroblasts. Proportioning: about 10 ten thousand porcine primary fibroblasts: 1 μ g YAP1-gRNA2:1 μ g YAP1-gRNA3: mu.g NCN protein. Co-transfection was performed by electroporation using a mammalian nuclear transfection kit (Neon kit, thermofeisher) and a Neon TM transfection system electrotransfer instrument (parameters set at 1450V, 10ms, 3 pulses).
2. After step 1, the culture is carried out for 16 to 18 hours by using the complete culture solution, and then the culture is carried out by replacing the complete culture solution with a new one. The total time of incubation after electroporation was 48 hours.
3. After completion of step 2, cells were trypsinized and collected, then washed with complete medium, then resuspended with complete medium, and then individual monoclonals were picked up and transferred to 96-well plates (1 cell per well with 100. Mu.l of complete medium per well) for 2 weeks (replacement of new complete medium every 2-3 days).
4. After completion of step 3, cells were trypsinized and harvested (approximately 2/3 of the resulting cells per well were plated into 6-well plates containing complete medium, and the remaining 1/3 were harvested in 1.5mL centrifuge tubes).
5. The 6-well plate of step 4 was taken, cultured until the cells grew to 80% confluency, trypsinized and collected, and the cells were cryopreserved using cell cryopreserving (90% complete medium +10% dmso by volume).
6. Taking the centrifuge tube in the step 4, taking the cells, performing cell lysis and extracting genomic DNA, performing PCR amplification by using a primer pair (the primer sequence is shown in example 4) consisting of YAP1-E2g-JDF132 and YAP1-E2g-JDR570, and performing electrophoresis. Porcine primary fibroblasts were used as wild type controls (WT).
7. After completion of step 6, the PCR amplification product was recovered and sequenced.
Sequencing of porcine primary fibroblasts resulted in only one genotype, wild type (also called homozygous wild type). If the sequencing result of a single-cell clone has two types, one type is consistent with the sequencing result of the pig primary fibroblast, and the other type has mutation (mutation comprises deletion, insertion or substitution of one or more nucleotides) compared with the sequencing result of the pig primary fibroblast, the genotype of the single-cell clone is heterozygote; if the sequencing result of one single-cell clone is two types, the two types of single-cell clones are mutated (the mutation comprises deletion, insertion or substitution of one or more nucleotides) compared with the sequencing result of the primary porcine fibroblast, and the genotype of the single-cell clone is a biallelic different mutant type; if the sequencing result of a single-cell clone is one and mutation (mutation comprises deletion, insertion or substitution of one or more nucleotides) is generated compared with the sequencing result of the pig primary fibroblast, the genotype of the single-cell clone is a biallelic identical mutant; if the sequencing result of a single cell clone is one and is consistent with the sequencing result of a pig primary fibroblast, the genotype of the single cell clone is a wild type (also called a homozygous wild type).
The results are shown in Table 1. The genotypes of the single cell clones numbered 4, 9, 13, 16, 20, 24, 34 were wild-type. The genotypes of the single cell clones numbered 2, 3, 8, 11, 15, 19, 23, 25, 29, 31, 35, 37, 41, 43 were heterozygous. The genotypes of the single cell clones numbered 1, 5, 7, 12, 14, 18, 22, 26, 28, 30, 32, 33, 36, 38, 40, 42 are biallelically distinct mutants. The genotypes of the single cell clones numbered 6, 10, 17, 21, 27, 39 were biallelic mutants. The rate of single cell clones resulting from YAP1 gene editing was 83.7%.
Exemplary sequencing alignments are shown in figures 3 to 6. FIG. 3 shows the alignment of the forward sequencing of clone No. 4 with the wild-type sequence, and the result was judged to be wild-type. FIG. 4 shows the result of alignment of the forward sequencing of clone No. 2 with the wild-type sequence, and it was judged as heterozygous. FIG. 5 shows the alignment of the forward and reverse sequencing of clone No. 1 with the wild type sequence, as biallelic different mutants. FIG. 6 is an alignment of the forward sequencing of clone No. 6 with the wild type sequence, showing a biallelic identity mutant.
TABLE 1 genotype determination results of single-cell clones edited by YAP1 Gene
Figure BDA0003221427390000101
Figure BDA0003221427390000111
Figure BDA0003221427390000121
The above described single cell clones of heterozygote type can be used for subsequent cloned pig production. The cells are used as nuclear transplantation donor cells to carry out somatic cell cloning, and cloned pigs, namely cataract model pigs, can be obtained.
Example 4 screening of efficient gRNA target of YAP1 Gene
Pig YAP1 gene information: encodes YES 1-related transcription factor 1; is located on chromosome 9; geneID is 100622418, sus scrofa. The amino acid sequence of the protein coded by the pig YAP1 gene is shown as SEQ ID NO: shown in fig. 8. In the porcine genomic DNA, the YAP1 gene has 9 exons, and a partial sequence (containing the No. 2 exon and the nucleotides of the upstream and downstream parts) thereof is shown as SEQ ID NO: shown at 9.
1. Conservation analysis of YAP1 gene default deletion region and adjacent genome sequence
18 newborn Jiangxiang pigs, wherein 10 females (named 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10) and 8 males (named A, B, C, D, E, F, G and H respectively) are provided.
YAP1-E2g-JDF50：ACTCATACTGTCTTGGCCTTTAC；
YAP1-E2g-JDR501：CATCATTATTTTCACTTACTTTA；
YAP1-E2g-JDF132：ATTTGTCTTTGGTATTTAGAGTT；
YAP1-E2g-JDR570：TTTATATTTTGGTACCACCTATG。
The porcine ear tissue designated 1 was used to extract the genome as a template, PCR amplified with different primer pairs, and then subjected to 1% agarose gel electrophoresis. The electrophoretogram is shown in FIG. 7. In fig. 7: group 1: adopting a primer pair consisting of YAP1-E2g-JDF50 and YAP1-E2g-JDR 570; group 2: adopting a primer pair consisting of YAP1-E2g-JDF50 and YAP1-E2g-JDR 501; group 3: adopting a primer pair consisting of YAP1-E2g-JDF132 and YAP1-E2g-JDR 570; group 4: a primer pair consisting of YAP1-E2g-JDF132 and YAP1-E2g-JDR501 was used. As a result, it is preferable to amplify the target fragment using a primer pair consisting of YAP1-E2g-JDF132 and YAP1-E2g-JDR 570.
The genomic DNA of 18 pigs was used as templates, and PCR amplification was performed using primer pairs consisting of YAP1-E2g-JDF132 and YAP1-E2g-JDR570, followed by 1% agarose gel electrophoresis. The electrophoretogram is shown in FIG. 8. And recovering PCR amplification products, sequencing, and comparing and analyzing the sequencing result with the YAP1 gene sequence in the public database. A common conserved region in 18 pigs is selected for designing a gRNA target.
2. Screening target spots
And (3) primarily screening a plurality of targets by screening NGG (avoiding possible mutation sites), and further screening 6 targets from the NGG through a preliminary experiment.
The 6 targets were as follows:
YAP1-E2-gRNA1：GCCAGAGACTACTCCAGTGG；
YAP1-E2-gRNA2：TGATGATGTACCTCTGCCAG；
YAP1-E2-gRNA3：AGATATGGCTCCCAGCTGCA；
YAP1-E2-gRNA4：TCTCAAAAGAAGACTGTCGA；
YAP1-E2-gRNA5：AGCTGGGAGCCATATCTCCT；
YAP1-E2-gRNA6：CTGTCGAAGGTGTTGAGCAG。
3. preparation of gRNA
The plasmid pKG-U6gRNA was digested with the restriction enzyme BbsI, and the vector backbone (approximately 3kb linear large fragment) was recovered.
YAP1-E2-gRNA1-S and YAP1-E2-gRNA1-A are synthesized respectively, and then mixed and annealed to obtain double-stranded DNA molecules with sticky ends. The double-stranded DNA molecule with the cohesive end was ligated to a vector backbone to obtain plasmid pKG-U6gRNA (YAP 1-E2-gRNA 1). Plasmid pKG-U6gRNA (YAP 1-E2-gRNA 1) expresses the nucleic acid sequence of SEQ ID NO:10 sgRNA YAP1-E2-gRNA1 。
sgRNA YAP1-E2-gRNA1 (SEQ ID NO：10)：
GCCAGAGACUACUCCAGUGGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu
YAP1-E2-gRNA2-S and YAP1-E2-gRNA2-A are synthesized respectively, and then mixed and annealed to obtain double-stranded DNA molecules with sticky ends. Double-stranded DNA molecules having cohesive ends were ligated to the vector backbone to obtain plasmid pKG-U6gRNA (YAP 1-E2-gRNA 2). Plasmid pKG-U6gRNA (YAP 1-E2-gRNA 2) expresses the nucleic acid sequence of SEQ ID NO:11 sgRNA YAP1-E2-gRNA2 。
sgRNA YAP1-E2-gRNA2 (SEQ ID NO：11)：
UGAUGAUGUACCUCUGCCAGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu
YAP1-E2-gRNA3-S and YAP1-E2-gRNA3-A are synthesized respectively, and then mixed and annealed to obtain double-stranded DNA molecules with sticky ends. Double-stranded DNA molecules having cohesive ends were ligated to the vector backbone to obtain plasmid pKG-U6gRNA (YAP 1-E2-gRNA 3). Plasmid pKG-U6gRNA (YAP 1-E2-gRNA 3) expresses the nucleic acid sequence of SEQ ID NO:12 sgRNA YAP1-E2-gRNA3 。
sgRNA YAP1-E2-gRNA3 (SEQ ID NO：12)：
AGAUAUGGCUCCCAGCUGCAguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu
Respectively synthesizing YAP1-E2-gRNA4-S and YAP1-E2-gRNA4-A, then mixing and annealing to obtain the double-stranded DNA molecule with a sticky end. The double-stranded DNA molecule with the cohesive end is connected with a vector framework to obtain a plasmid pKG-U6gRNA (YAP 1-E2-gRNA 4). Plasmid pKG-U6gRNA (YAP 1-E2-gRNA 4) expresses the nucleic acid sequence of SEQ ID NO:13 instituteShown sgRNA YAP1-E2-gRNA4 。
sgRNA YAP1-E2-gRNA4 (SEQ ID NO：13)：
UCUCAAAAGAAGACUGUCGAguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu
Respectively synthesizing YAP1-E2-gRNA5-S and YAP1-E2-gRNA5-A, then mixing and annealing to obtain the double-stranded DNA molecule with a sticky end. The double-stranded DNA molecule with the cohesive end is connected with a vector framework to obtain a plasmid pKG-U6gRNA (YAP 1-E2-gRNA 5). Plasmid pKG-U6gRNA (YAP 1-E2-gRNA 5) expresses the nucleic acid sequence of SEQ ID NO:14 sgRNA YAP1-E2-gRNA5 。
sgRNA YAP1-E2-gRNA5 (SEQ ID NO：14)：
AGCUGGGAGCCAUAUCUCCUguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu
YAP1-E2-gRNA6-S and YAP1-E2-gRNA6-A are synthesized respectively, and then mixed and annealed to obtain double-stranded DNA molecules with sticky ends. The double-stranded DNA molecule with the cohesive end is connected with a vector framework to obtain a plasmid pKG-U6gRNA (YAP 1-E2-gRNA 6). Plasmid pKG-U6gRNA (YAP 1-E2-gRNA 6) expresses the nucleic acid sequence of SEQ ID NO:15 sgRNA YAP1-E2-gRNA6 。
sgRNA YAP1-E2-gRNA6 (SEQ ID NO：15)：
CUGUCGAAGGUGUUGAGCAGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu
YAP1-E2-gRNA1-S：caccGCCAGAGACTACTCCAGTGG；
YAP1-E2-gRNA1-A：aaacCCACTGGAGTAGTCTCTGGC；
YAP1-E2-gRNA2-S：caccgTGATGATGTACCTCTGCCAG；
YAP1-E2-gRNA2-A：aaacCTGGCAGAGGTACATCATCAc；
YAP1-E2-gRNA3-S：caccgAGATATGGCTCCCAGCTGCA；
YAP1-E2-gRNA3-A：aaacTGCAGCTGGGAGCCATATCTc；
YAP1-E2-gRNA4-S：caccgTCTCAAAAGAAGACTGTCGA；
YAP1-E2-gRNA4-A：aaacTCGACAGTCTTCTTTTGAGAc；
YAP1-E2-gRNA5-S：caccgAGCTGGGAGCCATATCTCCT；
YAP1-E2-gRNA5-A：aaacAGGAGATATGGCTCCCAGCTc；
YAP1-E2-gRNA6-S：caccgCTGTCGAAGGTGTTGAGCAG；
YAP1-E2-gRNA6-A：aaacCTGCTCAACACCTTCGACAGc。
YAP1-E2-gRNA1-S, YAP1-E2-gRNA1-A, YAP1-E2-gRNA2-S, YAP1-E2-gRNA2-A, YAP1-E2-gRNA3-S, YAP1-E2-gRNA3-A, YAP1-E2-gRNA4-S, YAP1-E2-gRNA4-A, YAP1-E2-gRNA5-S, YAP1-E2-gRNA5-A, YAP1-E2-gRNA6-S and YAP1-E2-gRNA6-A are single-stranded DNA molecules.
4. Comparison of editing efficiency for different target combinations
1. Cotransfection
A first group: the plasmid pKG-U6gRNA (YAP 1-E2-gRNA 1) and the plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Proportioning: about 20 million porcine primary fibroblasts: 0.92 μ g plasmid pKG-U6gRNA (YAP 1-E2-gRNA 1): 1.08. Mu.g of plasmid pKG-GE3.
Second group: the plasmid pKG-U6gRNA (YAP 1-E2-gRNA 2) and the plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Proportioning: about 20 million porcine primary fibroblasts: 0.92. Mu.g of plasmid pKG-U6gRNA (YAP 1-E2-gRNA 2): 1.08. Mu.g of plasmid pKG-GE3.
Third group: the plasmid pKG-U6gRNA (YAP 1-E2-gRNA 3) and the plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Proportioning: about 20 million porcine primary fibroblasts: 0.92 μ g plasmid pKG-U6gRNA (YAP 1-E2-gRNA 3): 1.08. Mu.g of plasmid pKG-GE3.
And a fourth group: the plasmid pKG-U6gRNA (YAP 1-E2-gRNA 4) and the plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Proportioning: about 20 million porcine primary fibroblasts: 0.92 μ g plasmid pKG-U6gRNA (YAP 1-E2-gRNA 4): 1.08. Mu.g of plasmid pKG-GE3.
And a fifth group: the plasmid pKG-U6gRNA (YAP 1-E2-gRNA 5) and the plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Proportioning: about 20 million porcine primary fibroblasts: 0.92 μ g plasmid pKG-U6gRNA (YAP 1-E2-gRNA 5): 1.08. Mu.g of plasmid pKG-GE3.
A sixth group: the plasmid pKG-U6gRNA (YAP 1-E2-gRNA 6) and the plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Proportioning: about 20 ten thousand porcine primary fibroblasts: 0.92 μ g plasmid pKG-U6gRNA (YAP 1-E2-gRNA 6): 1.08. Mu.g of plasmid pKG-GE3.
A seventh group: the primary pig fibroblast is subjected to electrotransformation operation without adding plasmid under the same electrotransformation parameters.
Co-transfection was performed by electroporation using a mammalian nuclear transfection kit (Neon kit, thermofeisher) and a Neon TM transfection system electrotransfer instrument (parameters set at 1450V, 10ms, 3 pulses).
2. After step 1, the culture is carried out for 12 to 18 hours by using the complete culture solution, and then the culture is carried out by replacing the complete culture solution with a new one. The total time of incubation after electroporation was 48 hours.
3. After step 2 was completed, cells were digested and collected with trypsin, lysed, genomic DNA was extracted, PCR amplified using primer pairs consisting of YAP1-E2g-JDF132 and YAP1-E2g-JDR570, and then subjected to 1% agarose gel electrophoresis. And detecting the mutation of the target gene of the cell, and an electrophoretogram is shown in figure 9.
And cutting and recovering the target product, sending the target product to a sequencing company for sequencing, and analyzing a sequencing peak map by using a webpage version Synthego ICE tool to obtain the gene editing efficiency of different targets. The gene editing efficiencies of the first to sixth groups were 27%, 37%, 44%, 10%, 7%, and 13% in this order, and no gene editing occurred in the seventh group. The result shows that the editing efficiency of YAP1-E2-gRNA2 and YAP1-E2-gRNA3 is higher.
Example 5 Performance of NCN proteins
The 2 gRNA targets targeting the TTN gene were selected as follows:
TTN-gRNA1：AGAGCACAGTCAGCCTGGCG；
TTN-gRNA2：CTTCCAGAATTGGATCTCCG。
primers used to identify target fragments comprising grnas in the TTN gene were as follows:
TTN-F55：TACGGAATTGGGGAGCCAGCGGA；
TTN-R560：CAAAGTTAACTCTCTGTGTCT。
1. preparation of gRNA
1. Preparing TTN-T7-gRNA1 transcription template and TTN-T7-gRNA2 transcription template
The TTN-T7-gRNA1 transcription template is a double-stranded DNA molecule, and is shown as SEQ ID NO:4, respectively.
The TTN-T7-gRNA2 transcription template is a double-stranded DNA molecule, and is shown as SEQ ID NO:5, respectively.
2. In vitro transcription to obtain gRNA
Taking TTN-T7-gRNA1 Transcription template, adopting a Transcription Aid T7 High Yield Transcription Kit (Fermentas, K0441) to carry out in vitro Transcription, and then using MEGA clear TM The Transcription Clean-Up Kit (Thermo, AM 1908) was recovered and purified to obtain TTN-gRNA1.TTN-gRNA1 is a single-stranded RNA, as shown in SEQ ID NO: and 6.
Taking TTN-T7-gRNA2 Transcription template, adopting a Transcription Aid T7 High Yield Transcription Kit (Fermentas, K0441) to carry out in vitro Transcription, and then using MEGA clear TM The Transcription Clean-Up Kit (Thermo, AM 1908) was recovered and purified to obtain TTN-gRNA2.TTN-gRNA2 is single-stranded RNA, shown in SEQ ID NO: shown in fig. 7.
2. gRNA and NCN protein dosage proportion optimization
1. Co-transfected porcine primary fibroblasts
A first group: co-transfecting primary pig fibroblasts with TTN-gRNA1, TTN-gRNA2 and NCN protein. Proportioning: about 10 million porcine primary fibroblasts: 0.5 μ g TTN-gRNA1:0.5 μ g TTN-gRNA2: mu.g NCN protein.
Second group: co-transfecting the porcine primary fibroblasts with TTN-gRNA1, TTN-gRNA2 and NCN proteins. Proportioning: about 10 ten thousand porcine primary fibroblasts: 0.75 μ g TTN-gRNA1:0.75 μ g TTN-gRNA2: mu.g NCN protein.
Third group: co-transfecting the porcine primary fibroblasts with TTN-gRNA1, TTN-gRNA2 and NCN proteins. Proportioning: about 10 million porcine primary fibroblasts: 1 μ g TTN-gRNA1:1 μ g TTN-gRNA2: mu.g NCN protein.
And a fourth group: co-transfecting primary pig fibroblasts with TTN-gRNA1, TTN-gRNA2 and NCN protein. Proportioning: about 10 ten thousand porcine primary fibroblasts: 1.25 μ g TTN-gRNA1:1.25 μ g TTN-gRNA2: mu.g NCN protein.
And a fifth group: co-transfecting the porcine primary fibroblasts with TTN-gRNA1 and TTN-gRNA2. Proportioning: about 10 ten thousand porcine primary fibroblasts: 1 μ g TTN-gRNA1:1 μ g TTN-gRNA2.
Co-transfection was performed by electroporation using a mammalian nuclear transfection kit (Neon kit, thermofeisher) and a Neon TM transfection system electrotransfer instrument (parameters set at 1450V, 10ms, 3 pulses).
2. After the completion of step 1, the culture is carried out for 12 to 18 hours by using the complete culture solution, and then the culture is carried out by replacing with a new complete culture solution. The total time of incubation after electroporation was 48 hours.
3. After completion of step 2, cells were digested with trypsin and collected, genomic DNA was extracted, PCR amplified using a primer pair consisting of TTN-F55 and TTN-R560, and then subjected to 1% agarose gel electrophoresis.
The electrophoretogram is shown in FIG. 10. The 505bp band is wild type band (WT), and the about 254bp band (251 bp band is theoretically deleted from 505bp band of wild type) is deletion mutant band (MT).
Gene deletion mutation efficiency = (MT grayscale/MT band bp number)/(WT grayscale/WT band bp number + MT grayscale/MT band bp number) × 100%. The deletion mutation efficiency of the first group of genes is 19.9 percent, the deletion mutation efficiency of the second group of genes is 39.9 percent, the deletion mutation efficiency of the third group of genes is 79.9 percent, and the deletion mutation efficiency of the fourth group of genes is 44.3 percent. Group five was not mutated.
The result shows that when the mass ratio of the two gRNAs to the NCN protein is 1:1:4, the actual dosage is 1 mu g:1 μ g: the gene editing efficiency is highest at 4 mug. Thus, the optimal amount of two grnas and NCN proteins was determined to be 1 μ g:1 μ g:4 μ g.
3. Comparison of Gene editing efficiency of NCN protein with that of the commercial Cas9 protein
1. Co-transfected porcine primary fibroblasts
Cas9-a group: co-transfecting the TTN-gRNA1, the TTN-gRNA2 and a commercial Cas9-A protein into a pig primary fibroblast. Proportioning: about 10 million porcine primary fibroblasts: 1 μ g TTN-gRNA1:1 μ g TTN-gRNA2:4 μ g Cas9-A protein.
pKG-GE4 group: co-transfecting the porcine primary fibroblasts with TTN-gRNA1, TTN-gRNA2 and NCN proteins. Proportioning: about 10 million porcine primary fibroblasts: 1 μ g TTN-gRNA1:1 μ g TTN-gRNA2: mu.g NCN protein.
Cas9-B set: co-transfecting the TTN-gRNA1, the TTN-gRNA2 and a commercial Cas9-B protein into a pig primary fibroblast. Proportioning: about 10 million porcine primary fibroblasts: 1 μ g TTN-gRNA1:1 μ g TTN-gRNA2:4 μ g Cas9-B protein.
Control group: co-transfecting the TTN-gRNA1 and the TTN-gRNA2 to the pig primary fibroblasts. Proportioning: about 10 million porcine primary fibroblasts: 1 μ g TTN-gRNA1:1 μ g TTN-gRNA2.
Co-transfection was performed by electroporation using a mammalian nuclear transfection kit (Neon kit, thermofeisher) and a Neon TM transfection system electrotransfer instrument (parameters set at 1450V, 10ms, 3 pulses).
2. After the completion of step 1, the culture is carried out for 12 to 18 hours by using the complete culture solution, and then the culture is carried out by replacing with a new complete culture solution. The total time of incubation after electroporation was 48 hours.
3. After completion of step 2, cells were digested with trypsin and collected, genomic DNA was extracted, PCR amplified using a primer pair consisting of TTN-F55 and TTN-R560, and then subjected to 1% agarose gel electrophoresis.
The electrophoretogram is shown in FIG. 11. The gene deletion mutation efficiency with the commercial Cas9-a protein was 28.5%, the gene deletion mutation efficiency with the NCN protein was 85.6%, and the gene deletion mutation efficiency with the commercial Cas9-B protein was 16.6%.
The result shows that compared with the Cas9 protein which adopts a commodity, the NCN protein prepared by the invention can obviously improve the gene editing efficiency.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific examples, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is made possible within the scope of the claims attached below.
Sequence listing
<110> Nanjing King Gene engineering Co., ltd
<120> CRISPR system for constructing cataract model pig nuclear transplantation donor cells with YAP1 gene mutation and application thereof
<130> GNCYX212444
<160> 19
<170> SIPOSequenceListing 1.0
<210> 1
<211> 9974
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60
cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120
ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180
gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240
acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300
ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360
ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420
acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480
tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540
tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600
gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 660
ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720
agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780
agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840
tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900
tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960
cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020
aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080
tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140
tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200
ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260
ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320
cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380
gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440
actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500
aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560
caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620
aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680
accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740
aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800
ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860
agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920
accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980
gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040
tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100
cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160
cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220
cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280
ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340
taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400
gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460
tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520
cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580
gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640
gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700
catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760
tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820
ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880
tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940
ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000
aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060
gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120
tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180
acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3240
cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300
cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg 3360
gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc 3420
cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg 3480
gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca 3540
tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag 3600
atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt 3660
tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 3720
gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc 3780
tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc 3840
cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct 3900
tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc ggactcggta 3960
atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg 4020
atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct 4080
tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga 4140
cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc 4200
aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg 4260
ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct 4320
tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt 4380
tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc 4440
gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc 4500
gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc 4560
gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact 4620
ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga 4680
taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc 4740
ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg 4800
atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag 4860
tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc 4920
gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat 4980
gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc 5040
aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat 5100
cgatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa 5160
ttcccctcta gaaataattt tgtttaactt taagaaggag atatacatat gaaacaaagc 5220
actattgcac tggcactctt accgttactg tttacccctg tgacaaaagc catgagcgat 5280
aaaattattc acctgactga cgacagtttt gacacggatg tactcaaagc ggacggggcg 5340
atcctcgtcg atttctgggc agagtggtgc ggtccgtgca aaatgatcgc cccgattctg 5400
gatgaaatcg ctgacgaata tcagggcaaa ctgaccgttg caaaactgaa catcgatcaa 5460
aaccctggca ctgcgccgaa atatggcatc cgtggtatcc cgactctgct gctgttcaaa 5520
aacggtgaag tggcggcaac caaagtgggt gcactgtcta aaggtcagtt gaaagagttc 5580
ctcgacgcta acctggccgg ttctggttct ggccatatgc accatcatca tcatcatgac 5640
gatgacgata agatgcccaa aaagaaacga aaggtgggta tccacggagt cccagcagcc 5700
gacaaaaaat atagcatcgg cctggacatc ggtaccaaca gcgttggctg ggcagtgatc 5760
actgatgaat acaaagttcc atccaaaaaa tttaaagtac tgggcaacac cgaccgtcac 5820
tctatcaaaa aaaacctgat tggtgctctg ctgtttgaca gcggcgaaac tgctgaggct 5880
acccgtctga aacgtacggc tcgccgtcgc tacactcgtc gtaaaaaccg catctgttat 5940
ctgcaggaaa ttttctctaa cgaaatggca aaagttgatg atagcttctt tcatcgtctg 6000
gaagagagct tcctggtgga agaagataaa aaacacgaac gtcacccgat tttcggtaac 6060
attgtggatg aggttgccta ccacgagaaa tatccgacca tctaccatct gcgtaaaaaa 6120
ctggttgata gcactgacaa agcggatctg cgtctgatct acctggctct ggcacacatg 6180
atcaaattcc gtggtcactt cctgatcgaa ggtgatctga accctgataa ctccgacgtg 6240
gacaaactgt tcattcagct ggttcagacc tataaccagc tgttcgaaga aaacccgatc 6300
aacgcgtccg gtgtagacgc taaggcaatt ctgtctgcgc gtctgtctaa gtctcgtcgt 6360
ctggaaaacc tgattgcgca actgccaggt gaaaagaaaa acggcctgtt cggcaatctg 6420
atcgccctgt ccctgggtct gactccgaac tttaaatcca actttgacct ggcggaagat 6480
gccaagctgc agctgagcaa agatacctat gacgatgacc tggataacct gctggcacag 6540
atcggtgatc agtatgccga tctgttcctg gccgcgaaaa acctgtctga tgcgattctg 6600
ctgtctgata tcctgcgcgt taacactgaa attactaaag cgccgctgag cgcatccatg 6660
attaaacgtt acgatgaaca ccaccaggat ctgaccctgc tgaaagcgct ggtgcgtcag 6720
cagctgccgg aaaaatacaa ggagatcttc ttcgaccaga gcaaaaacgg ttacgcgggc 6780
tacattgatg gtggtgcatc tcaggaggaa ttctacaaat tcattaaacc gatcctggaa 6840
aaaatggatg gtactgaaga gctgctggtt aaactgaatc gtgaagatct gctgcgcaaa 6900
cagcgtacct tcgataacgg ttccatcccg catcagattc atctgggcga actgcacgct 6960
atcctgcgcc gtcaggaaga cttttatccg ttcctgaaag acaaccgtga gaaaattgaa 7020
aaaatcctga ccttccgtat tccgtactat gtaggtccgc tggcgcgtgg taactcccgt 7080
ttcgcttgga tgacccgcaa aagcgaagaa accatcaccc cgtggaattt cgaagaagtc 7140
gttgacaaag gcgcgtccgc gcagtctttc atcgaacgca tgacgaactt cgacaaaaac 7200
ctgccgaacg agaaagtgct gccgaaacac tctctgctgt acgagtactt cactgtgtac 7260
aacgaactga ccaaagtgaa atacgtcacc gaaggtatgc gtaaaccggc attcctgtcc 7320
ggtgagcaaa aaaaagcaat cgtggatctg ctgttcaaaa ccaaccgtaa agtaaccgtg 7380
aaacagctga aggaagacta tttcaagaaa atcgaatgtt ttgattctgt tgaaatctcc 7440
ggcgtggaag atcgcttcaa tgcgtccctg ggtacgtatc acgacctgct gaaaattatc 7500
aaagacaaag attttctgga caacgaggaa aacgaagaca tcctggagga tattgtactg 7560
accctgaccc tgttcgaaga ccgtgagatg atcgaagaac gcctgaaaac ctacgcccac 7620
ctgttcgatg acaaggtaat gaagcagctg aaacgtcgtc gttataccgg ctggggtcgt 7680
ctgtcccgta aactgatcaa tggcatccgt gataaacagt ctggcaaaac catcctggac 7740
ttcctgaaat ccgacggttt cgcgaatcgt aacttcatgc aactgattca tgacgattct 7800
ctgactttca aagaagacat ccagaaagca caggtttccg gccagggtga ctctctgcac 7860
gagcacattg ccaatctggc tggttctccg gctattaaaa agggtattct gcagactgtg 7920
aaagtagttg atgagctggt caaagtaatg ggccgtcaca agccggaaaa cattgtgatc 7980
gaaatggcac gtgaaaacca gacgacccag aaaggtcaga aaaactctcg tgaacgcatg 8040
aaacgtatcg aagaaggcat caaagaactg ggctctcaga tcctgaagga acaccctgta 8100
gaaaataccc agctgcagaa cgaaaagctg tatctgtatt acctgcagaa cggccgcgat 8160
atgtatgtgg accaggaact ggatatcaac cgcctgtccg attacgatgt agatcacatc 8220
gtgccgcaaa gcttcctgaa agacgacagc attgacaaca aagtactgac ccgttctgat 8280
aagaaccgtg gcaaatccga taacgtcccg tctgaagaag ttgttaaaaa aatgaaaaac 8340
tattggcgtc agctgctgaa cgcgaaactg atcacccagc gtaagttcga caatctgact 8400
aaagctgagc gcggtggtct gtccgaactg gataaagcgg gttttatcaa acgccagctg 8460
gttgaaaccc gtcagatcac gaagcacgtt gcgcagattc tggactctcg tatgaacacc 8520
aaatacgacg aaaacgacaa actgatccgc gaggttaagg ttatcaccct gaaaagcaaa 8580
ctggtatccg attttcgtaa agactttcag ttctacaaag tgcgcgaaat taacaactat 8640
caccacgctc acgatgcata tctgaatgca gttgttggca cggcgctgat caaaaagtat 8700
ccgaaactgg aatctgaatt cgtatacggc gattacaaag tgtatgacgt tcgtaagatg 8760
atcgcaaaat ccgagcagga aattggtaag gcgacggcga aatacttctt ttattccaat 8820
attatgaact ttttcaaaac cgaaatcacc ctggcgaatg gtgaaattcg taaacgcccg 8880
ctgatcgaaa ccaacggtga aactggtgaa atcgtttggg acaaaggccg cgacttcgcg 8940
accgtgcgta aagttctgtc tatgccgcaa gtgaacatcg tcaagaagac cgaagtacaa 9000
accggcggtt ttagcaaaga gagcattctg ccaaaacgta actccgacaa actgatcgcg 9060
cgcaagaaag actgggatcc gaaaaaatac ggtggtttcg attctccaac cgttgcttat 9120
tccgttctgg tggtagccaa agttgagaaa ggtaaaagca aaaaactgaa atccgtaaag 9180
gaactgctgg gtattactat catggagcgt agctccttcg aaaaaaaccc gatcgatttt 9240
ctggaagcga aaggctataa agaagtcaaa aaggacctga tcatcaaact gccaaaatac 9300
agcctgttcg agctggaaaa cggccgtaaa cgtatgctgg catctgcggg cgaactgcag 9360
aaaggcaacg agctggctct gccgtccaaa tacgtgaact ttctgtacct ggcctctcac 9420
tacgaaaaac tgaaaggttc cccggaagac aacgaacaga aacagctgtt cgtagagcag 9480
cacaaacact acctggacga gatcatcgaa cagatttctg aattttctaa acgtgtgatt 9540
ctggctgatg cgaatctgga taaagttctg tctgcctata acaagcatcg tgacaaaccg 9600
atccgcgaac aggctgagaa catcatccac ctgttcactc tgactaacct gggcgcgcca 9660
gcggctttca agtactttga taccaccatt gaccgcaagc gttacacctc cactaaagaa 9720
gtgctggacg cgactctgat ccaccagtcc atcaccggtc tgtacgagac ccgtatcgat 9780
ctgagccagc tgggcggtga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa 9840
aagaaaaagt gacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata 9900
actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg 9960
aactatatcc ggat 9974
<210> 2
<211> 1547
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Met Lys Gln Ser Thr Ile Ala Leu Ala Leu Leu Pro Leu Leu Phe Thr
1 5 10 15
Pro Val Thr Lys Ala Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp
20 25 30
Ser Phe Asp Thr Asp Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp
35 40 45
Phe Trp Ala Glu Trp Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu
50 55 60
Asp Glu Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu
65 70 75 80
Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly
85 90 95
Ile Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys
100 105 110
Val Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn
115 120 125
Leu Ala Gly Ser Gly Ser Gly His Met His His His His His His Asp
130 135 140
Asp Asp Asp Lys Met Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly
145 150 155 160
Val Pro Ala Ala Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr
165 170 175
Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser
180 185 190
Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys
195 200 205
Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala
210 215 220
Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn
225 230 235 240
Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val
245 250 255
Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu
260 265 270
Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu
275 280 285
Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys
290 295 300
Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala
305 310 315 320
Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp
325 330 335
Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val
340 345 350
Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly
355 360 365
Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg
370 375 380
Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu
385 390 395 400
Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys
405 410 415
Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp
420 425 430
Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln
435 440 445
Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu
450 455 460
Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu
465 470 475 480
Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr
485 490 495
Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu
500 505 510
Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly
515 520 525
Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu
530 535 540
Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp
545 550 555 560
Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln
565 570 575
Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe
580 585 590
Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr
595 600 605
Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg
610 615 620
Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn
625 630 635 640
Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu
645 650 655
Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro
660 665 670
Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr
675 680 685
Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser
690 695 700
Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg
705 710 715 720
Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu
725 730 735
Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala
740 745 750
Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp
755 760 765
Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu
770 775 780
Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys
785 790 795 800
Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg
805 810 815
Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly
820 825 830
Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser
835 840 845
Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser
850 855 860
Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly
865 870 875 880
Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile
885 890 895
Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys
900 905 910
Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg
915 920 925
Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met
930 935 940
Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys
945 950 955 960
Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu
965 970 975
Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp
980 985 990
Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser
995 1000 1005
Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp
1010 1015 1020
Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys
1025 1030 1035 1040
Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr
1045 1050 1055
Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser
1060 1065 1070
Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg
1075 1080 1085
Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr
1090 1095 1100
Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr
1105 1110 1115 1120
Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr
1125 1130 1135
Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu
1140 1145 1150
Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu
1155 1160 1165
Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met
1170 1175 1180
Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe
1185 1190 1195 1200
Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala
1205 1210 1215
Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr
1220 1225 1230
Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys
1235 1240 1245
Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln
1250 1255 1260
Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp
1265 1270 1275 1280
Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly
1285 1290 1295
Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val
1300 1305 1310
Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly
1315 1320 1325
Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe
1330 1335 1340
Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys
1345 1350 1355 1360
Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met
1365 1370 1375
Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro
1380 1385 1390
Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu
1395 1400 1405
Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln
1410 1415 1420
His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser
1425 1430 1435 1440
Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala
1445 1450 1455
Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile
1460 1465 1470
Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys
1475 1480 1485
Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu
1490 1495 1500
Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu
1505 1510 1515 1520
Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Lys Arg Pro Ala Ala
1525 1530 1535
Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys
1540 1545
<210> 3
<211> 1399
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Met Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala Ala
1 5 10 15
Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly
20 25 30
Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys
35 40 45
Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly
50 55 60
Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys
65 70 75 80
Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr
85 90 95
Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe
100 105 110
Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His
115 120 125
Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His
130 135 140
Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser
145 150 155 160
Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met
165 170 175
Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp
180 185 190
Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn
195 200 205
Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys
210 215 220
Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu
225 230 235 240
Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu
245 250 255
Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp
260 265 270
Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp
275 280 285
Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu
290 295 300
Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile
305 310 315 320
Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met
325 330 335
Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala
340 345 350
Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp
355 360 365
Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln
370 375 380
Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly
385 390 395 400
Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys
405 410 415
Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly
420 425 430
Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu
435 440 445
Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro
450 455 460
Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met
465 470 475 480
Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val
485 490 495
Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn
500 505 510
Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu
515 520 525
Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr
530 535 540
Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys
545 550 555 560
Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val
565 570 575
Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser
580 585 590
Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr
595 600 605
Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn
610 615 620
Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu
625 630 635 640
Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His
645 650 655
Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
660 665 670
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys
675 680 685
Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala
690 695 700
Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys
705 710 715 720
Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His
725 730 735
Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile
740 745 750
Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg
755 760 765
His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr
770 775 780
Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu
785 790 795 800
Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val
805 810 815
Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln
820 825 830
Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu
835 840 845
Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp
850 855 860
Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly
865 870 875 880
Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn
885 890 895
Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
900 905 910
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys
915 920 925
Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys
930 935 940
His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu
945 950 955 960
Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
965 970 975
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu
980 985 990
Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val
995 1000 1005
Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val
1010 1015 1020
Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser
1025 1030 1035 1040
Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn
1045 1050 1055
Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile
1060 1065 1070
Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val
1075 1080 1085
Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met
1090 1095 1100
Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe
1105 1110 1115 1120
Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala
1125 1130 1135
Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro
1140 1145 1150
Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys
1155 1160 1165
Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met
1170 1175 1180
Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys
1185 1190 1195 1200
Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr
1205 1210 1215
Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala
1220 1225 1230
Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val
1235 1240 1245
Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro
1250 1255 1260
Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr
1265 1270 1275 1280
Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile
1285 1290 1295
Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His
1300 1305 1310
Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe
1315 1320 1325
Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr
1330 1335 1340
Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala
1345 1350 1355 1360
Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp
1365 1370 1375
Leu Ser Gln Leu Gly Gly Asp Lys Arg Pro Ala Ala Thr Lys Lys Ala
1380 1385 1390
Gly Gln Ala Lys Lys Lys Lys
1395
<210> 4
<211> 225
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ggcttgtcgg actcttcgct attacgccag ctggcgaagg gggatgtgct gcaaggcgat 60
taagttgggt aacgccaggg ttttcccagt cacgacgtta ggaaattaat acgactcact 120
ataggagagc acagtcagcc tggcggtttt agagctagaa atagcaagtt aaaataaggc 180
tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttt 225
<210> 5
<211> 225
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ggcttgtcgg actcttcgct attacgccag ctggcgaagg gggatgtgct gcaaggcgat 60
taagttgggt aacgccaggg ttttcccagt cacgacgtta ggaaattaat acgactcact 120
ataggcttcc agaattggat ctccggtttt agagctagaa atagcaagtt aaaataaggc 180
tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttt 225
<210> 6
<211> 102
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ggagagcaca gucagccugg cgguuuuaga gcuagaaaua gcaaguuaaa auaaggcuag 60
uccguuauca acuugaaaaa guggcaccga gucggugcuu uu 102
<210> 7
<211> 102
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
ggcuuccaga auuggaucuc cgguuuuaga gcuagaaaua gcaaguuaaa auaaggcuag 60
uccguuauca acuugaaaaa guggcaccga gucggugcuu uu 102
<210> 8
<211> 505
<212> PRT
<213> Sus scrofa
<400> 8
Met Asp Pro Gly Gln Gln Gln Pro Pro Pro Gln Pro Ala Pro Gln Gly
1 5 10 15
Gln Gly Gln Pro Pro Ala Gln Pro Pro Gln Gly Gln Gly Pro Pro Ser
20 25 30
Gly Pro Gly Gln Pro Ala Pro Pro Gly Ser Gln Ala Ala Thr Gln Xaa
35 40 45
Pro Pro Ala Gly His Gln Ile Val His Val Arg Gly Asp Ser Glu Thr
50 55 60
Asp Leu Glu Ala Leu Phe Asn Ala Val Met Asn Pro Lys Thr Ala Asn
65 70 75 80
Val Pro Gln Thr Val Pro Met Arg Leu Arg Lys Leu Pro Asp Ser Phe
85 90 95
Phe Lys Pro Pro Glu Pro Lys Ser His Ser Arg Gln Ala Ser Thr Asp
100 105 110
Ala Gly Thr Ala Gly Ala Leu Thr Pro Gln His Val Arg Ala His Ser
115 120 125
Ser Pro Ala Ser Leu Gln Leu Gly Ala Ile Ser Pro Gly Thr Leu Thr
130 135 140
Pro Thr Gly Val Val Ser Gly Pro Ala Ala Thr Pro Thr Ala Gln His
145 150 155 160
Leu Arg Gln Ser Ser Phe Glu Ile Pro Asp Asp Val Pro Leu Pro Ala
165 170 175
Gly Trp Glu Met Ala Lys Thr Ser Ser Gly Gln Arg Tyr Phe Leu Asn
180 185 190
His Ile Asp Gln Thr Thr Thr Trp Gln Asp Pro Arg Lys Ala Met Leu
195 200 205
Ser Gln Met Asn Val Thr Ala Pro Thr Ser Pro Pro Val Gln Gln Asn
210 215 220
Met Met Asn Ser Ala Ser Gly Pro Leu Pro Asp Gly Trp Glu Gln Ala
225 230 235 240
Met Thr Gln Asp Gly Glu Ile Tyr Tyr Ile Asn His Lys Asn Lys Thr
245 250 255
Thr Ser Trp Leu Asp Pro Arg Leu Asp Pro Arg Phe Ala Met Asn Gln
260 265 270
Arg Ile Ser Gln Ser Ala Pro Val Lys Gln Pro Pro Pro Leu Ala Pro
275 280 285
Gln Ser Pro Gln Gly Gly Val Met Gly Gly Gly Ser Ser Asn Gln Gln
290 295 300
Gln Gln Met Arg Leu Gln Gln Leu Gln Met Glu Lys Glu Arg Leu Arg
305 310 315 320
Leu Lys Gln Gln Glu Leu Leu Arg Gln Ala Met Arg Asn Ile Asn Pro
325 330 335
Ser Thr Ala Asn Ser Pro Lys Cys Gln Glu Leu Ala Leu Arg Ser Gln
340 345 350
Leu Pro Thr Leu Glu Gln Asp Gly Gly Thr Gln Asn Pro Val Ser Ser
355 360 365
Pro Gly Met Ser Gln Glu Leu Arg Thr Met Thr Thr Asn Ser Ser Asp
370 375 380
Pro Phe Leu Asn Ser Gly Thr Tyr His Ser Arg Asp Glu Ser Thr Asp
385 390 395 400
Ser Gly Leu Ser Met Ser Ser Tyr Ser Val Pro Arg Thr Pro Asp Asp
405 410 415
Phe Leu Asn Ser Val Asp Glu Met Asp Thr Gly Asp Thr Ile Asn Gln
420 425 430
Ser Thr Leu Pro Ser Gln Gln Asn Arg Phe Pro Asp Tyr Leu Glu Ala
435 440 445
Ile Pro Gly Thr Asn Val Asp Leu Gly Thr Leu Glu Gly Asp Gly Met
450 455 460
Asn Ile Glu Gly Glu Glu Leu Met Pro Ser Leu Gln Glu Ala Leu Ser
465 470 475 480
Ser Asp Ile Leu Asn Asp Met Glu Ser Val Leu Ala Ala Thr Lys Leu
485 490 495
Asp Lys Glu Ser Phe Leu Thr Trp Leu
500 505
<210> 9
<211> 951
<212> DNA
<213> Sus scrofa
<400> 9
acatatggat ggttttggat cttttgtatt cgttaaaagt tatgttaatt ttggtggttg 60
ctttattgat gacatttgtc cgtaatgggg gtaaaaaatg actaccatca tatgtttagg 120
gcttggcttg ggaaaaatat tacttaaaac ctagtttaaa gcgtagttat atcccttggg 180
ttttgtttat acccagaaca ttttagaatt tggaaaagca ctcatactgt cttggccttt 240
acataagcaa agtatccatc tgcaaatcta tatacctctt tgtcaaatac taaatgctga 300
aatttgtctt tggtatttag agtttcactg caaccaaata tctaaggaat ttaaagtgaa 360
aacaattttt aagaaatttt cattttaata ttcctaatag gccagtactg atgcaggcac 420
tgcaggggcc ctgactccac agcatgttcg agctcattcc tctccagctt ccctgcagct 480
gggagccata tctcctggga cactgacccc cactggagta gtctctggcc cagcagctac 540
gcccactgct caacaccttc gacagtcttc ttttgagata cctgatgatg tacctctgcc 600
agcgggctgg gagatggcca aaacatcttc tggtcagaga tacttcttaa agtaagtgaa 660
aataatgatg ggtgatggtt ttctgagggg aaaaaaaaaa aacctatttt ggaaaacata 720
ggtggtacca aaatataaaa tgtcatttgc ttttatattt tacttaatat ttacatcaaa 780
ctcagttatg tcttctgtaa cttttttttc agtttaataa gacctaaata ccttttttgg 840
ataatagttt aaaagcttta ttgatgcctc gttcatagcc ttttaataat cttttgtgcc 900
aactttggat atatgtttca aacctattaa ttaacttagc tcataaggaa a 951
<210> 10
<211> 100
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
gccagagacu acuccagugg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 11
<211> 100
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
ugaugaugua ccucugccag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 12
<211> 100
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
agauauggcu cccagcugca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 13
<211> 100
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
ucucaaaaga agacugucga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 14
<211> 100
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
agcugggagc cauaucuccu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 15
<211> 100
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
cugucgaagg uguugagcag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 16
<211> 225
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
ggcttgtcgg actcttcgct attacgccag ctggcgaagg gggatgtgct gcaaggcgat 60
taagttgggt aacgccaggg ttttcccagt cacgacgtta ggaaattaat acgactcact 120
ataggtgatg atgtacctct gccaggtttt agagctagaa atagcaagtt aaaataaggc 180
tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttt 225
<210> 17
<211> 225
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
ggcttgtcgg actcttcgct attacgccag ctggcgaagg gggatgtgct gcaaggcgat 60
taagttgggt aacgccaggg ttttcccagt cacgacgtta ggaaattaat acgactcact 120
ataggagata tggctcccag ctgcagtttt agagctagaa atagcaagtt aaaataaggc 180
tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttt 225
<210> 18
<211> 102
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
ggugaugaug uaccucugcc agguuuuaga gcuagaaaua gcaaguuaaa auaaggcuag 60
uccguuauca acuugaaaaa guggcaccga gucggugcuu uu 102
<210> 19
<211> 102
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
ggagauaugg cucccagcug caguuuuaga gcuagaaaua gcaaguuaaa auaaggcuag 60
uccguuauca acuugaaaaa guggcaccga gucggugcuu uu 102

Claims (14)

  1. Application of YAP1-gRNA2, YAP1-gRNA3 and NCN protein in preparation of a kit;
    the YAP1-gRNA2 is sgRNA, and a target sequence binding region of the YAP1-gRNA is shown in SEQ ID NO:18, nucleotides 3 to 22; the YAP1-gRNA3 is sgRNA, and a target sequence binding region is shown in SEQ ID NO:19 at nucleotides 3 to 22; the NCN protein is a Cas9 protein or a fusion protein with a Cas9 protein;
    the application of the kit is as follows (a), (b) or (c): (a) preparing a recombinant cell; (b) preparing cataract model pig; (c) Preparing a cataract cell model or a cataract tissue model or a cataract organ model.
  2. 2, application of YAP1-gRNA2, YAP1-gRNA3 and PRONCN protein in preparation of a kit;
    YAP1-gRNA2 is YAP1-gRNA2 as described in claim 1; YAP1-gRNA3 is YAP1-gRNA3 as described in claim 1; the PRONCN protein sequentially comprises the following elements from upstream to downstream: signal peptide, molecular chaperone protein, protein tag, protease enzyme cutting site, nuclear localization signal, cas9 protein and nuclear localization signal;
    the application of the kit is as follows (a), (b) or (c): (a) preparing a recombinant cell; (b) preparing cataract model pig; (c) Preparing a cataract cell model or a cataract tissue model or a cataract organ model.
  3. The application of YAP1-gRNA2, YAP1-gRNA3 and the very-heterogeneous particles in the preparation of the kit;
    YAP1-gRNA2 is YAP1-gRNA2 as defined in claim 1; YAP1-gRNA3 is YAP1-gRNA3 as described in claim 1; the specific plasmid sequentially comprises the following elements from upstream to downstream: a promoter, an operator, a ribosome binding site, a PRONCN protein coding gene and a terminator; the PRONCN protein sequentially comprises the following elements from upstream to downstream: signal peptide, molecular chaperone protein, protein tag, protease enzyme cutting site, nuclear localization signal, cas9 protein and nuclear localization signal;
    the application of the kit is as follows (a), (b) or (c): (a) preparing a recombinant cell; (b) preparing cataract model pig; (c) Preparing a cataract cell model or a cataract tissue model or a cataract organ model.
  4. 4. A method of making a recombinant cell comprising the steps of: co-transfecting a pig cell with YAP1-gRNA2, YAP1-gRNA3 and NCN protein to obtain a recombinant cell; YAP1-gRNA2 is YAP1-gRNA2 as defined in claim 1; YAP1-gRNA3 is YAP1-gRNA3 as described in claim 1; the NCN protein is the NCN protein according to claim 1.
  5. 5. The method of claim 4, wherein: the proportions of the pig cells, YAP1-gRNA2, YAP1-gRNA3 and NCN protein are as follows: 10 ten thousand porcine cells: 0.8-1.2 μ g YAP1-gRNA2:0.8-1.2 μ g YAP1-gRNA3: 3-5. Mu.g NCN protein.
  6. 6. Recombinant cells produced by the method of claim 4 or 5.
  7. 7. Use of the recombinant cell of claim 6 for the preparation of cataract-model pigs.
  8. 8. Porcine tissue, organ or cell of cataract-model pig prepared by using the recombinant cell of claim 6.
  9. 9. The recombinant cell of claim 6, the porcine tissue of claim 8, the porcine organ of claim 8, the porcine cell of claim 8, or the cataract model pig prepared by using the recombinant cell of claim 6, wherein the recombinant cell is (d 1) or (d 2) or (d 3) or (d 4):
    (d1) Screening the medicine for treating cataract;
    (d2) Evaluating the drug effect of the cataract drug;
    (d3) Evaluating the curative effect of gene therapy and/or cell therapy of cataract;
    (d4) The pathogenesis of cataract is studied.
  10. 10. A kit comprising YAP1-gRNA2, YAP1-gRNA3, and NCN protein;
    YAP1-gRNA2 is YAP1-gRNA2 as described in claim 1; YAP1-gRNA3 is YAP1-gRNA3 as described in claim 1; the NCN protein is the NCN protein described in claim 1;
    the application of the kit is as follows (a), (b) or (c): (a) preparing a recombinant cell; (b) preparing cataract model pig; (c) Preparing a cataract cell model or a cataract tissue model or a cataract organ model.
  11. 11. A kit comprising YAP1-gRNA2, YAP1-gRNA3 and PRONCN protein;
    YAP1-gRNA2 is YAP1-gRNA2 as defined in claim 1; YAP1-gRNA3 is YAP1-gRNA3 as described in claim 1; the PRONCN protein is the PRONCN protein of claim 2;
    the application of the kit is as follows (a), (b) or (c): (a) preparing a recombinant cell; (b) preparing cataract model pig; (c) Preparing a cataract cell model or a cataract tissue model or a cataract organ model.
  12. 12. A kit comprises YAP1-gRNA2, YAP1-gRNA3 and specific plasmids;
    YAP1-gRNA2 is YAP1-gRNA2 as described in claim 1; YAP1-gRNA3 is YAP1-gRNA3 as described in claim 1; the specific plasmid is the specific plasmid described in claim 3;
    the application of the kit is as follows (a), (b) or (c): (a) preparing a recombinant cell; (b) preparing cataract model pig; (c) Preparing a cataract cell model or a cataract tissue model or a cataract organ model.
  13. 13. The use according to claim 1 or the method according to claim 4 or 5 or the kit according to claim 10, characterized in that: the NCN protein is shown as SEQ ID NO:3, respectively.
  14. 14. The use or method or kit as claimed in claim 13, wherein:
    the preparation method of the NCN protein comprises the following steps:
    (1) Introducing the plasmid pKG-GE4 into escherichia coli BL21 (DE 3) to obtain recombinant bacteria;
    (2) Culturing the recombinant strain by adopting a liquid culture medium at 30 ℃, then adding IPTG (isopropyl-beta-D-thiogalactoside) and carrying out induced culture at 25 ℃, and then collecting the strain;
    (3) Crushing the collected thalli, and collecting a crude protein solution;
    (4) Purification of the crude protein solution with His by affinity chromatography 6 A fusion protein of the tag;
    (5) By using a compound having His 6 Tagged enterokinase cleavage with His 6 Tag fusion protein, then removing His in the protein with Ni-NTA resin 6 A tagged protein, resulting in a purified NCN protein;
    plasmid pKG-GE4 has the sequence SEQ ID NO:1, nucleotide 5209-9852.
CN202110959122.3A 2021-08-20 2021-08-20 CRISPR system for constructing cataract model pig nuclear transplantation donor cells with YAP1 gene mutation and application thereof Pending CN115232816A (en)

Priority Applications (1)

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CN202110959122.3A CN115232816A (en) 2021-08-20 2021-08-20 CRISPR system for constructing cataract model pig nuclear transplantation donor cells with YAP1 gene mutation and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110959122.3A CN115232816A (en) 2021-08-20 2021-08-20 CRISPR system for constructing cataract model pig nuclear transplantation donor cells with YAP1 gene mutation and application thereof

Publications (1)

Publication Number Publication Date
CN115232816A true CN115232816A (en) 2022-10-25

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Country Link
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