CN110004145B - sgRNA, knockout vector, knockout method of KLF4 gene and application thereof - Google Patents
sgRNA, knockout vector, knockout method of KLF4 gene and application thereof Download PDFInfo
- Publication number
- CN110004145B CN110004145B CN201910263714.4A CN201910263714A CN110004145B CN 110004145 B CN110004145 B CN 110004145B CN 201910263714 A CN201910263714 A CN 201910263714A CN 110004145 B CN110004145 B CN 110004145B
- Authority
- CN
- China
- Prior art keywords
- sequence
- klf4 gene
- sgrna
- knockout
- vector
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Mycology (AREA)
- Cell Biology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a sgRNA, a double-stranded DNA, a vector and a cell. The invention also discloses a method for knocking out the KLF4 gene. The invention firstly utilizes CRISPR/Cas9 technology to construct a KLF4 knockout IPEC-J2 cell line which can be used as an ideal cell model for pig disease research; the gene knockout is carried out by using the CRISPR/Cas9 technology, and the KLF4 gene can be knocked out more effectively than technical means such as gene silencing and interference, and the like, thereby being more beneficial to the function research of the KLF4 protein; gene sequencing detection shows that the KLF4 gene sequence is knocked out, so that the function of the KLF4 gene can be deleted, and the IPEC-J2 cell model is an ideal KLF4 gene knock-out IPEC-J2 cell model. The knockout method is simple and convenient, and the targeting efficiency is high.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to an application technology of CRISPR/Cas9, a pig small intestine epithelial cell line (IPEC-J2) gene knockout vector, a gene knockout method and application thereof.
Background
The CRISPR/Cas9 technology is an adaptive immune system mediated by sgRNA (small guide RNA) from one of bacteria and archaea. Wherein the sgRNA is used for identifying and combining target DNA, and can combine and cut DNA at the downstream of a region after the activity of the Cas9 protein is activated to generate double-stranded DNA break, induce frame shift mutation manufactured by a non-homologous end connection repair machine, and further realize the editing of a target gene. Due to the characteristics of simplicity and high efficiency of the CRISPR/Cas9 technology, the CRISPR/Cas9 technology is widely applied to the field of research on gene functions of human beings, animals and plants as an important genetic means for fixed-point editing of eukaryotic genes in recent years.
The porcine small intestinal epithelial cell (IPEC-J2) is separated from the columnar epithelial cell in the middle section of jejunum of a newborn piglet, has species specificity for researching porcine pathogen infection, is an ideal in-vitro model for researching various diseases of the pig, and is particularly widely applied to the identification research of enterotoxigenic escherichia coli and viral diarrhea resistance gene functions.
KLF4 is a member of Kruppel-like transcription factor family, participates in important life processes such as regulation of cell proliferation, differentiation and embryonic development, and mediates innate immune response. At present, no porcine KLF4 gene knockout vector or a porcine KLF4 gene knockout small intestinal epithelial cell model exists.
Disclosure of Invention
The purpose of the invention is as follows: in order to overcome the defects in the prior art, the invention provides an sgRNA.
The technical problem to be solved by the invention is to provide a double-stranded DNA.
The technical problem to be solved by the invention is to provide a vector or a cell.
The invention finally solves the technical problem of providing a knockout method of the KLF4 gene.
The technical scheme is as follows: in order to solve the technical problems, the technical scheme adopted by the invention is as follows: an sgRNA having a base sequence of ACAGCCATGTCAGACTCGCC.
The invention further comprises a double-stranded DNA, wherein the upstream sequence of the double-stranded DNA is as follows:
sgRNA3F：
the downstream sequence of the double-stranded DNA is:
sgRNA3R：
the invention further discloses a vector containing the sgRNA or the double-stranded DNA, and the vector is a CRISPR/Cas9 knockout vector capable of targeting pig KLF4 genes.
The invention also relates to a cell comprising said vector.
The invention also comprises a method for knocking out the KLF4 gene, which comprises the following steps:
1) Target site design and sgRNA sequence synthesis: finding out the 5' end of a CDs region according to a pig KLF4 gene to knock out a target site and designing an sgRNA guide sequence;
2) Construction of targeting vectors: annealing the designed sgRNA guide sequence to form a double-stranded DNA sequence, then connecting the double-stranded DNA sequence with a vector to obtain a connecting product, introducing the connecting product into escherichia coli to obtain a bacterial colony, extracting plasmids from the bacterial colony to obtain a knockout vector, designing a PCR primer according to the knockout vector and the sgRNA sequence, and further sequencing and verifying the PCR product of the bacterial colony to obtain a positive targeting vector;
3) Cell transfection: adding the positive targeting vector into a suspension of IPEC-J2 cells in a logarithmic growth phase for electrotransformation to obtain a cell sap;
4) Knock-out of KLF4 gene: and (2) screening puromycin from cell sap, transferring and expanding the obtained monoclonal cells, separating out partial cells and extracting genomic DNA of the cells when the cells are overgrown, designing a high-specificity primer near a knockout target site according to a KLF4 gene complete sequence, carrying out PCR amplification on the extracted genomic DNA to obtain a PCR product, and verifying the product.
Wherein, the PCR primer in the step 2) is:
F3：
R3：
wherein the reaction program of colony PCR in the step 2) is 95 ℃ for 10min; 35sec at 95 ℃, 35sec at 60 ℃, 35sec at 72 ℃,35 cycles; 10min at 72 ℃.
Wherein the IPEC-J2 cell concentration in the step 3) is 1-2 multiplied by 10 6 One per ml.
Wherein, the primer sequence with high specificity in the step 4) is as follows:
F：
R：
wherein, the PCR reaction program of the step 4) is 95 ℃ for 5min;95 ℃ 10sec,60 ℃ 10sec,72 ℃ 10sec,35 cycles; 5min at 72 ℃.
The knockout method specifically comprises the following steps:
1) Adding sgRNA sequence of CACCG to 5' end
And annealing the reverse complementary sequence of the sgRNA sequence with AAAC added at the 5' end to form double-stranded DNA;
2) Connecting the double-stranded DNA with a linearized knockout vector pGK2.1 to obtain a connection product;
3) The primer is used for:
F1：
R1：
F2：
R2：
F3：
R3：
performing colony PCR screening on the ligation product, and obtaining a positive targeting vector through amplification culture and plasmid extraction;
4) Transfecting the IPEC-J2 cell with the positive targeting vector, extracting cell genome DNA, designing a high-specificity primer near a knockout target site, and performing PCR amplification;
5)Cruiser TM screening enzyme digestion PCR products to obtain 1 sgRNA capable of efficiently knocking out KLF4 gene;
6) And (5) carrying out sequencing verification on the PCR product to obtain the IPEC-J2 cell with the KLF4 gene knocked out.
According to the invention, the CRISPR/Cas9 technology is adopted to knock out the KLF4 gene sequence in the IPEC-J2 cell line genome, and the established IPEC-J2 cell knocked out by the KLF4 gene can provide a more direct and effective research model for deeply disclosing the action mechanism of the KLF4 gene in disease resistance and the application of the KLF4 gene in pig disease resistance breeding.
Has the advantages that: compared with the prior art, the invention has the following advantages:
1. the IPEC-J2 cell line with KLF4 knockout is constructed by using a CRISPR/Cas9 technology for the first time, and can be used as an ideal cell model for pig disease research;
2. the gene knockout is carried out by using the CRISPR/Cas9 technology, and compared with the technical means such as gene silencing, interference and the like, the gene knockout of the KLF4 gene can be more effective, and the functional research of the KLF4 protein is more facilitated;
3. gene sequencing detection shows that the KLF4 gene sequence is knocked out, so that the function of the KLF4 gene can be deleted, and the IPEC-J2 cell model is an ideal KLF4 gene knock-out IPEC-J2 cell model.
4. The knockout method is simple and convenient, and the provided sgRNA sequence can realize high-efficiency knockout of the target gene.
Drawings
FIG. 1 is a diagram of colony PCR gel electrophoresis;
FIG. 2 is a colony PCR product sequencing peak diagram: fig. 2A is a diagram of sequencing peaks of sgRNA1 corresponding to the targeting vector; fig. 2B is a graph of sequencing peaks of sgRNA2 corresponding to the targeting vector; fig. 2C is a graph of sequencing peaks of sgRNA3 corresponding to the targeting vector;
FIG. 3 is a gel electrophoresis assay of PCR amplification of knock-out target sites;
FIG. 4 is a Cruiser TM Performing enzyme digestion and screening on a DNASPCR product gel electrophoresis detection map of the KLF4 gene knockout cell;
FIG. 5 is a Cruiser TM Carrying out enzyme digestion and screening on a DNAPR product sequencing peak map of the KLF4 gene knockout cell;
FIG. 6 shows the result of comparative analysis of the DNA sequence of KLF4 gene knock-out cells and the DNA sequence of KLF4 gene non-knock-out cells.
Detailed Description
The present invention will be further described with reference to the accompanying drawings.
Example 1
1. Target site design and sgRNA sequence synthesis:
obtaining a transcript CDs sequence of a porcine KLF4 Gene (Gene ID: 595111) according to an NCBI database, finding out the 5' end of a CDs region for knocking out a target site Design, and designing three sgRNA guide sequences by using CRISPR Design (http:// CRISPR. Mit. Edu /):
in three sgRNA guide sequences sgRNA1:
sgRNA2：
sgRNA3：
adding CACCG at the 5' end of the gene to form a plus chain sgRNA DNA sequence; simultaneously synthesizing a reverse complementary sequence of a plus-strand sgRNA DNA sequence: sgRNAl':
sgRNA2’：
sgRNA3’：
AAAC was added to the 5' end to form a minus strand sgRNA DNA sequence.
Three pairs of sgRNA sequences are as follows:
sgRNA1F：
sgRNA1R：
sgRNA2F：
sgRNA2R：
sgRNA3F：
sgRNA3R：
2. construction of targeting vectors:
(1) Annealing the positive and negative strand sgRNA sequences to form a double-stranded DNA sequence, wherein the reaction system is as follows:
the reaction system is centrifuged instantly and incubated at 95 ℃ for 3min, and then cooled naturally for 20min.
(2) The enzyme digestion linearization is carried out on the knocking-out vector pGK2.1 by BbsI enzyme, and the reaction system is as follows:
and (4) incubating at 37 ℃ for 4h, and purifying and recovering the linearized vector pGK2.1 by using the kit.
(3) Connecting the double-stranded DNA sequence formed by annealing with a linearized vector pGK2.1, wherein the reaction system is as follows:
the above system was centrifuged instantaneously and incubated at 16 ℃ for 30min to obtain a ligation product.
(4) Adding more than 10 mu l of ligation product into DH5 alpha competence, gently mixing uniformly, and incubating on ice for 30min; thermally shocking for 60sec at 42 ℃, quickly taking out, placing on ice and cooling for 2-3 min; adding 800 μ l of nonreactive SOC liquid culture medium into the tube, and culturing for 45min with shaking table (37 deg.C/160 rpm); centrifugation was carried out at 4500rpm for 5min, 600. Mu.l of the supernatant was discarded, and the pellet was resuspended in the remaining 200. Mu.l of the supernatant, spread evenly on kanamycin-resistant culture plates, and cultured overnight at 37 ℃ while being inverted.
Three pairs of PCR primers are designed according to the knockout vector pGK2.1 and three sgRNA sequences:
F1：
R1：
F2：
R2：
F3：
R3：
screening of colony PCR: white monoclonal colonies were picked from the above plates and placed in a PCR tube, and 1. Mu.l each of the above upstream and downstream primers, 2. Mu.l of buffer (10X), 2. Mu.l of dNTP, 0.2. Mu.l of Taq enzyme, and 13.8. Mu.l of double distilled water were added to the PCR tube. The PCR reaction program is 95 ℃ for 10min; 35sec at 95 ℃, 35sec at 60 ℃, 35sec at 72 ℃,35 cycles; 10min at 72 ℃.
FIG. 1 is a PCR gel electrophoresis test chart of colonies, wherein M is 100bp DNA marker, and 1, 2 and 3 are PCR bands of positive colonies. As can be seen from fig. 1: the positive targeting vector was obtained in the above manner.
The obtained PCR product of the positive colony is further sequenced and verified, and as shown in figure 2, the gene sequence of the positive targeting vector is identical with the pGK2.1 vector and the sgRNA sequence. And performing amplification culture on the positive targeting vector by adopting a conventional method, and performing plasmid extraction for subsequent experiments.
3. Cell transfection:
about 2X 10 of log phase growth 6 Each porcine small intestinal epithelial cell (IPEC-J2) was placed in a 15mL centrifuge tube, centrifuged at 1000rpm/5min to discard the supernatant, the cells were suspended with 210. Mu.l DPBS, and transferred to a 1.5mL centrifuge tube. Respectively adding 5-8 mu g of the 3 targeting carriers into the 1.5mL centrifuge tube containing the IPEC-J2 cell suspension liquid, gently mixing uniformly, transferring the mixed liquid into an electric shock cup by using a special electric transfer gun head, covering an electric shock cup cover after the liquid level bulges, placing the electric shock cup cover on an electric transfer instrument, carrying out electric transfer at 650V/30ms, and transferring the cell sap into a six-hole plateIn a culture medium.
Knock-out of KLF4 gene:
after 72 hours of electroporation, the cells were screened with puromycin at a concentration of 3. Mu.g/ml and diluted by limiting dilution into 10 96 well plates in CO at 37 ℃ in a standard format 2 After standing culture in the incubator for 7 days, growth of the monoclonal cells was observed, and after about 14 days, the monoclonal cells were transferred to 48 wells for scale-up culture. When the cells grew over 48 wells, a part of the cells were isolated and their genomic DNA was extracted using a DNA extraction kit.
PCR amplification of the DNA extracted in step 4:
designing a high-specificity primer near a knockout target site according to the KLF4 gene complete sequence, wherein the sequence is as follows:
F：
R：
the PCR amplification reaction system is as follows:
the PCR reaction program is 95 ℃ for 5min;95 ℃ for 10sec,60 ℃ for 10sec,72 ℃ for 10sec,35 cycles; a PCR product of length 326 bp was obtained at 72 ℃ 5min,95 ℃ 3 min.
FIG. 3 is a gel electrophoresis assay of PCR amplification of knock-out target sites. Wherein M is 100bp DNA marker, and 1, 2 and 3 are PCR product bands. As can be seen from fig. 3: the primer designed near the target site can be knocked out to successfully amplify the target sequence.
Screening and verifying KLF4 gene knockout cells:
(1)Cruiser TM enzyme digestion screening of KLF4 gene knockout cells
Using a Cruiser recognizing mismatches TM Enzyme-cutting and detecting the PCR product with the length of 326 bp to prepare a reaction systemThe following were used:
after 20min reaction at 45 ℃ and 2. Mu.l of 6 XStop Buffer was added to the 10. Mu.l reaction system, agarose gel electrophoresis was performed. FIG. 4 is a Cruiser TM Performing enzyme digestion screening on a KLF4 gene knockout cell result, wherein M represents 100bpDNAmarker;1-1, 1-2 represent cells transfected with sgRNA1 targeting vectors; 2-1, 2-2 represent cells transfected with sgRNA2 targeting vectors; 3-1, 3-2 represent cells transfected with sgRNA3 targeting vectors. As can be seen from fig. 4, sgRNA1 and sgRNA2 failed to efficiently knock out the KLF4 gene, while sgRNA3 efficiently knocked out the KLF4 gene. The results of the analysis of the targeting efficiency based on the gray scale values showed that the targeting efficiency of KLF4 gene was 54.8% and 56.1% in the cells corresponding to 3-1 and 3-2, respectively.
(2) DNA PCR product sequencing verification of KLF4 gene knockout cells:
for Cruiser TM Sequencing verification is carried out on the KLF4 gene knockout cell DNACR product obtained by enzyme digestion screening, and as shown in a sequencing peak diagram of FIG. 5, the cell transfected with the sgRNA3 targeting vector is determined to be the KLF4 gene knockout cell. The DNA of the KLF4 gene knockout cell is subjected to TA cloning and then sequenced, and is compared with the DNA sequence of the KLF4 gene knockout cell, and the DNA sequence of the knockout cell is mutated into deletion of 116bp and 137bp (figure 6).
FIG. 6 shows the sequence of KLF4 gene knockout by TA clone sequencing analysis. Wherein, the first row is a sequence without a KLF4 gene knockout, and the second row is a KLF4 gene knockout sequence.
The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.
Sequence listing
<110> Yangzhou university
<120> sgRNA, knockout vector, knockout method of KLF4 gene and application thereof
<160> 17
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> sgRNA1 (Artificial Sequence)
<400> 1
acgttattcg gggcacctgc 20
<210> 2
<211> 19
<212> DNA
<213> sgRNA2(Artificial Sequence)
<400> 2
acaacactca cgttattcg 19
<210> 3
<211> 20
<212> DNA
<213> sgRNA3(Artificial Sequence)
<400> 3
acagccatgt cagactcgcc 20
<210> 4
<211> 24
<212> DNA
<213> F1(Artificial Sequence)
<400> 4
catatgctta ccgtaacttg aaag 24
<210> 5
<211> 21
<212> DNA
<213> R1(Artificial Sequence)
<400> 5
gcaggtgccc cgaataacgt c 21
<210> 6
<211> 24
<212> DNA
<213> F2(Artificial Sequence)
<400> 6
catatgctta ccgtaacttg aaag 24
<210> 7
<211> 19
<212> DNA
<213> R2(Artificial Sequence)
<400> 7
cgaataacgt gagtgttgt 19
<210> 8
<211> 24
<212> DNA
<213> F3(Artificial Sequence)
<400> 8
catatgctta ccgtaacttg aaag 24
<210> 9
<211> 20
<212> DNA
<213> R3(Artificial Sequence)
<400> 9
ggcgagtctg acatggctgt 20
<210> 10
<211> 25
<212> DNA
<213> sgRNA1F(Artificial Sequence)
<400> 10
caccgacgtt attcggggca cctgc 25
<210> 11
<211> 25
<212> DNA
<213> sgRNA1R(Artificial Sequence)
<400> 11
aaacgcaggt gccccgaata acgtc 25
<210> 12
<211> 24
<212> DNA
<213> sgRNA2F(Artificial Sequence)
<400> 12
caccgacaac actcacgtta ttcg 24
<210> 13
<211> 23
<212> DNA
<213> sgRNA2R(Artificial Sequence)
<400> 13
aaaccgaata acgtgagtgt tgt 23
<210> 14
<211> 25
<212> DNA
<213> sgRNA3F(Artificial Sequence)
<400> 14
caccgacagc catgtcagac tcgcc 25
<210> 15
<211> 24
<212> DNA
<213> sgRNA3R(Artificial Sequence)
<400> 15
aaacggcgag tctgacatgg ctgt 24
<210> 16
<211> 20
<212> DNA
<213> F(Artificial Sequence)
<400> 16
ggacttcgga ggacctctga 20
<210> 17
<211> 20
<212> DNA
<213> R(Artificial Sequence)
<400> 17
ccacccacag aagcccacta 20
Claims (10)
1. An sgRNA having a base sequence of ACAGCCATGTCAGACTCGCC.
2. A double-stranded DNA having the upstream sequence: 5'-CACCGACAGCCATGTCAGACTCGCC-3', the downstream sequence of the double stranded DNA is: sgRNA3R: 5'-AAACGGCGAGTCTGACATGGCTGT-3'.
3. A vector comprising the sgRNA of claim 1 or the double-stranded DNA of claim 2.
4. A cell comprising the vector of claim 3.
5. A method for knocking out a KLF4 gene, comprising the steps of:
1) Target site design and sgRNA sequence synthesis: finding out the 5' end of a CDs region according to a pig KLF4 gene to knock out a target site and designing an sgRNA guide sequence; the sgRNA guide sequence is ACAGCCATGTCAGACTCGCC;
2) Construction of targeting vectors: annealing the designed sgRNA guide sequence to form a double-stranded DNA sequence, connecting the double-stranded DNA sequence with a vector to obtain a connecting product, introducing the connecting product into escherichia coli to obtain a bacterial colony, extracting plasmids from the bacterial colony to obtain a knockout vector, designing a PCR primer according to the knockout vector and the sgRNA sequence, and further sequencing and verifying the PCR product of the bacterial colony to obtain a positive targeting vector;
3) Cell transfection: adding the positive targeting vector into a suspension of IPEC-J2 cells in a logarithmic growth phase for electrotransformation to obtain a cell sap;
4) Knock-out of KLF4 gene: and (2) screening puromycin from cell sap, transferring and expanding the obtained monoclonal cells, separating out partial cells and extracting genomic DNA of the cells when the cells are overgrown, designing a high-specificity primer near a knockout target site according to a KLF4 gene complete sequence, carrying out PCR amplification on the extracted genomic DNA to obtain a PCR product, and verifying the product.
6. The KLF4 gene knock-out method of claim 5, wherein the PCR primers in step 2) are: f3:5'-CATATGCTTACCGTAACTTGAAAG-3'; r3:5'-GGCGAGTCTGACATGGCTGT-3'.
7. The KLF4 gene knock-out method according to claim 5, wherein the reaction program of colony PCR in step 2) is 95 ℃ for 10min; 35sec at 95 ℃,35 sec at 60 ℃,35 sec at 72 ℃,35 cycles; 10min at 72 ℃.
8. The KLF4 gene knock-out method according to claim 5, wherein the IPEC-J2 cell concentration in step 3) is 1~2 x 106 cells/ml.
9. The KLF4 gene knock-out method according to claim 5, wherein the primer sequence with high specificity in step 4) is: 5'-GGACTTCGGAGGACCTCTGA-3'; r is 5'-CCACCCACAGAAGCCCACTA-3'.
10. The KLF4 gene knock-out method according to claim 5, wherein the PCR reaction sequence of step 4) is 95 ℃ for 5min;95 ℃ 10sec,60 ℃ 10sec,72 ℃ 10sec,35 cycles; 5min at 72 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910263714.4A CN110004145B (en) | 2019-04-02 | 2019-04-02 | sgRNA, knockout vector, knockout method of KLF4 gene and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910263714.4A CN110004145B (en) | 2019-04-02 | 2019-04-02 | sgRNA, knockout vector, knockout method of KLF4 gene and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110004145A CN110004145A (en) | 2019-07-12 |
| CN110004145B true CN110004145B (en) | 2023-01-31 |
Family
ID=67169534
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910263714.4A Active CN110004145B (en) | 2019-04-02 | 2019-04-02 | sgRNA, knockout vector, knockout method of KLF4 gene and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110004145B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112011539A (en) * | 2020-08-26 | 2020-12-01 | 扬州大学 | Cell line for targeted knockout of porcine GDPD2 gene based on CRISPR-Cas9 technology and construction method thereof |
| CN112553200A (en) * | 2020-12-07 | 2021-03-26 | 扬州大学 | Preparation method and application of targeting vector for knocking out pig UGT2C1 gene |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107245498A (en) * | 2017-06-30 | 2017-10-13 | 浙江大学 | The construction method and purposes of pig pCRTC3 sgRNA expression vectors |
| CN108148861A (en) * | 2018-01-17 | 2018-06-12 | 扬州大学 | A kind of the HEK293 cell lines and its construction method of RNA methylases TRDMT1 gene knockouts |
| CN108220338A (en) * | 2017-11-22 | 2018-06-29 | 扬州大学 | A kind of construction method of the IPEC-J2 cells of APN gene knockouts |
| CN108265116A (en) * | 2018-01-22 | 2018-07-10 | 上海市第人民医院 | Klf4 is as liver cancer diseases diagnose and treat target spot |
| CN108949832A (en) * | 2018-07-13 | 2018-12-07 | 中国农业大学 | A kind of targeting vector and its application for knock-out pig GHR gene |
-
2019
- 2019-04-02 CN CN201910263714.4A patent/CN110004145B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107245498A (en) * | 2017-06-30 | 2017-10-13 | 浙江大学 | The construction method and purposes of pig pCRTC3 sgRNA expression vectors |
| CN108220338A (en) * | 2017-11-22 | 2018-06-29 | 扬州大学 | A kind of construction method of the IPEC-J2 cells of APN gene knockouts |
| CN108148861A (en) * | 2018-01-17 | 2018-06-12 | 扬州大学 | A kind of the HEK293 cell lines and its construction method of RNA methylases TRDMT1 gene knockouts |
| CN108265116A (en) * | 2018-01-22 | 2018-07-10 | 上海市第人民医院 | Klf4 is as liver cancer diseases diagnose and treat target spot |
| CN108949832A (en) * | 2018-07-13 | 2018-12-07 | 中国农业大学 | A kind of targeting vector and its application for knock-out pig GHR gene |
Non-Patent Citations (4)
| Title |
|---|
| 张廷焕等.猪MITF-M的转录调控分析.《畜牧兽医学报》.2018,(第11期), * |
| 杨红文 等."猪Klf4、Klf5、Klf7和Egr2基因的克隆及分子特性研究".《中国博士学位论文全文数据库（电子期刊）基础科学辑》.2008,(第02期), * |
| 杨红文.猪Klf4、Klf5、Klf7和Egr2基因扩增及在前脂肪细胞3T3-L1中的表达.《江苏农业学报》.2010,(第06期), * |
| 赵荣荣等.用CRISPR/Cas9技术编辑KLF4基因对GES-1细胞生物学行为的影响.《安徽医科大学学报》.2017,(第10期), * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110004145A (en) | 2019-07-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111607594B (en) | CRISPR-Cas9 editing technology-based cell line for knocking out pig IRF8 gene and construction method thereof | |
| CN106701830B (en) | Pig embryo p66 knock-outshcMethod for gene | |
| CN105671080B (en) | Method for sheep MSTN gene knockout and site-specific integration exogenous gene mediated by CRISPR-Cas9 system | |
| CN113278619B (en) | Double sgRNA, gene knockout vector, pig fibroblast line with STING gene knockout function and construction method thereof | |
| CN107418974A (en) | It is a kind of to sort the quick method for obtaining CRISPR/Cas9 gene knockout stable cell lines using monoclonal cell | |
| CN106755091A (en) | Gene knockout carrier, MH7A cell NLRP1 gene knockout methods | |
| CN104109687A (en) | Construction and application of Zymomonas mobilis CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-association proteins)9 system | |
| CN109750035B (en) | sgRNA for targeting and guiding Cas9 protein to efficiently cleave TCR and B2M gene locus | |
| CN111849979B (en) | sgRNA for targeted knockout of RPSA gene and construction method of RPSA gene knockout cell line | |
| CN110819592A (en) | Universal donor stem cell and preparation method thereof | |
| CN110004145B (en) | sgRNA, knockout vector, knockout method of KLF4 gene and application thereof | |
| CN112011539A (en) | Cell line for targeted knockout of porcine GDPD2 gene based on CRISPR-Cas9 technology and construction method thereof | |
| CN111041029A (en) | Strong promoter and application thereof in production of vitamin B12Application in bacterial strains | |
| CN112029699B (en) | Clostridium butyricum gene editing system based on endogenous CRISPR-Cas system and application thereof | |
| CN110184302B (en) | Construction method of SIRT1 gene knockout IPEC-J2 cell line | |
| CN114107304B (en) | Recombinant coccidium vector for expressing alpha toxin protein and fluorescent tag protein and detection method thereof | |
| CN104388456A (en) | Construction method of vector capable of simultaneously expressing two sgRNAs | |
| CN110591993A (en) | Vibrio harveyi homologous recombination gene knockout method based on hydrochloric acid stimulation | |
| CN112553200A (en) | Preparation method and application of targeting vector for knocking out pig UGT2C1 gene | |
| CN115948402A (en) | Recombinant Shewanella capable of producing 5-aminolevulinic acid and application thereof | |
| CN112921040A (en) | Construction method of pig TFF1 gene knockout cell line based on CRISPR-Cas9 gene editing technology | |
| JP2023106944A (en) | Primer set which specifically detects lactic acid bacteria lq80 strain, kit including primer set, and detection method using primer set | |
| CN114107299A (en) | sgRNA for targeted duck cGAS gene knockout and application thereof | |
| CN108754019B (en) | Amplification method of porcine epidemic diarrhea virus ORF1 gene complete sequence | |
| CN113684209A (en) | sgRNA capable of effectively editing pig PCBP1 gene and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |