CN110004145B - A kind of knockout method of sgRNA, knockout carrier, KLF4 gene and application thereof - Google Patents

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

A kind of knockout method of sgRNA, knockout carrier, KLF4 gene and application thereof

technical field

The invention belongs to the technical field of genetic engineering, and specifically relates to the application technology of CRISPR/Cas9, and also relates to a gene knockout vector of a porcine intestinal epithelial cell line (IPEC-J2), a gene knockout method and applications thereof.

Background technique

CRISPR/Cas9 technology is an adaptive immune system mediated by sgRNA (small guide RNA) derived from bacteria and archaea. Among them, sgRNA is used to recognize and bind target DNA. When the Cas9 protein activity is activated, it can bind to the DNA downstream of the region and cut it, generating double-strand DNA breaks and inducing frameshift mutations caused by non-homologous end-joining repair mechanisms. , so as to realize the editing of the target gene. Due to its simplicity and high efficiency, CRISPR/Cas9 technology has been widely used in the field of gene function research in humans, animals and plants in recent years as an important genetic method for site-specific editing of eukaryotic genes.

Porcine intestinal epithelial cells (IPEC-J2) are isolated from the columnar epithelial cells in the middle section of the jejunum of newborn piglets. They have species-specificity for the study of porcine pathogen infection and are ideal in vitro models for the study of various diseases in pigs, especially in enterotoxigenic Escherichia coli and It is widely used in the functional identification of viral diarrhea resistance genes.

KLF4 is a member of the Kruppel-like transcription factor family, which is involved in the regulation of important life processes such as cell proliferation, differentiation, and embryonic development, and mediates innate immune responses. At present, there are no pig KLF4 gene knockout vectors and KLF4 gene knockout small intestinal epithelial cell models.

Contents of the invention

Purpose of the invention: In order to overcome the deficiencies in the prior art, the technical problem to be solved by the present invention is to provide a sgRNA.

The technical problem to be solved by the present invention is to provide a double-stranded DNA.

The technical problem to be solved by the present invention is to provide a carrier or cell.

The final technical problem to be solved by the present invention is to provide a KLF4 gene knockout method.

Technical solution: In order to solve the above technical problems, the technical solution adopted in the present invention is: a sgRNA, the base sequence of which is ACAGCCATGTCAGACTCGCC.

The content of the present invention also includes a double-stranded DNA, the upstream sequence of the double-stranded DNA is:

sgRNA3F:

The downstream sequence of double-stranded DNA is:

sgRNA3R:

The content of the present invention also includes the carrier of the sgRNA or the double-stranded DNA, which is a CRISPR/Cas9 knockout carrier capable of targeting the porcine KLF4 gene.

The content of the present invention also includes cells of the vector.

The content of the present invention also includes a KLF4 gene knockout method, comprising the following steps:

1) Target site design and sgRNA sequence synthesis: find out the 5' end of the CDs region according to the porcine KLF4 gene to knock out the target site and design the sgRNA guide sequence;

2) Construction of the targeting vector: anneal the designed sgRNA guide sequence to form a double-stranded DNA sequence, and then connect it with the carrier to obtain a ligation product, introduce the ligation product into E. coli to obtain a colony, and extract the plasmid from the colony to obtain a knockout vector. and sgRNA sequence to design PCR primers, and further sequence and verify the colony PCR products to obtain positive targeting vectors;

3) Cell transfection: adding the positive targeting vector to the suspension of IPEC-J2 cells in the logarithmic growth phase for electroporation to obtain cell fluid;

4) KLF4 gene knockout: Puromycin was used to screen the cell fluid for drugs, and the obtained monoclonal cells were transferred and expanded for culture. When the cells were full, some cells were isolated and their genomic DNA was extracted. According to the complete sequence of the KLF4 gene , design highly specific primers near the knockout target site, and then perform PCR amplification on the extracted genomic DNA to obtain PCR products, and then verify the products.

Wherein, the PCR primers in the step 2) are:

F3:

R3:

Wherein, the reaction program of the colony PCR in the step 2) is 95° C. for 10 min; 95° C. for 35 sec, 60° C. for 35 sec, 72° C. for 35 sec, 35 cycles; 72° C. for 10 min.

Wherein, the IPEC-J2 cell concentration in step 3) is 1-2×10 ⁶ cells/ml.

Wherein, the step 4) highly specific primer sequence is:

F:

R:

Wherein, the step 4) PCR reaction program is 95° C. for 5 min; 95° C. for 10 sec, 60° C. for 10 sec, 72° C. for 10 sec, 35 cycles; 72° C. for 5 min.

The knockout method of the present invention specifically comprises the following steps:

和5’端添加AAAC的sgRNA序列的反向互补序列退火形成双链DNA；1) Add the sgRNA sequence of CACCG to the 5' end

Anneal to the reverse complementary sequence of the sgRNA sequence with AAAC added at the 5' end to form double-stranded DNA;

2) Ligate the double-stranded DNA with the linearized knockout vector pGK2.1 to obtain a ligation product;

3) With primers:

F1:

R1:

F2:

R2:

F3:

R3:

Colony PCR screening was carried out on the ligation products, and positive targeting vectors were obtained after expanding culture and plasmid extraction;

4) Transfect the positive targeting vector into IPEC-J2 cells, extract the genomic DNA of the cells, design highly specific primers near the knockout target site, and perform PCR amplification;

5) One sgRNA that can efficiently knock out the KLF4 gene was obtained by screening the PCR products digested by Cruiser ^TM ;

6) The PCR product was sequenced and verified to obtain KLF4 gene knockout IPEC-J2 cells.

The present invention uses CRISPR/Cas9 technology to knock out the KLF4 gene sequence in the genome of IPEC-J2 cell line, and the established KLF4 gene knockout IPEC-J2 cells can deeply reveal the mechanism of action of KLF4 gene in disease resistance and its role in pig resistance. The application in disease breeding provides a more direct and effective research model.

Beneficial effect: compared with the prior art, the present invention has the following advantages:

1. For the first time, the KLF4 knockout IPEC-J2 cell line was constructed using CRISPR/Cas9 technology, which can be used as an ideal cell model for pig disease research;

2. The use of CRISPR/Cas9 technology for gene knockout can knock out the KLF4 gene more effectively than gene silencing, interference and other technical means, and is more conducive to the functional research of KLF4 protein;

3. Gene sequencing test shows that knockout of KLF4 gene sequence can cause loss of KLF4 gene function, which is an ideal KLF4 gene knockout IPEC-J2 cell model.

4. The knockout method of the present invention is simple and convenient, and the provided sgRNA sequence can realize high-efficiency knockout of the target gene.

Description of drawings

Fig. 1 is colony PCR gel electrophoresis detection figure;

Figure 2 is the peak map of colony PCR product sequencing: Figure 2A is the peak map of the sequencing corresponding to the targeting carrier of sgRNA1; Figure 2B is the peak map of the sequencing corresponding to the targeting carrier of sgRNA2; Figure 2C is the peak map of the sequencing corresponding to the targeting carrier of sgRNA3;

Fig. 3 is the gel electrophoresis detection figure of knockout target site PCR amplification;

Fig. 4 is the gel electrophoresis detection diagram of DNA PCR products of KLF4 gene knockout cells screened by Cruiser ^TM enzyme digestion;

Figure 5 is the sequencing peak diagram of DNA PCR products of KLF4 gene knockout cells screened by Cruiser ^TM enzyme digestion;

Figure 6 is the result of comparative analysis of DNA sequences of KLF4 gene knockout cells and DNA sequences of KLF4 gene knockout cells.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings.

Example 1

1. Target site design and sgRNA sequence synthesis:

According to the NCBI database, the transcript CDs sequence of the porcine KLF4 gene (Gene ID: 595111) was obtained, and the 5' end of the CDs region was identified for knockout target site design, using CRISPR Design (http://crispr.mit.edu/) Three sgRNA guide sequences were designed:

sgRNA2：

sgRNA3：

在其5’端添加CACCG，形成正链sgRNA DNA序列；同时合成正链sgRNA DNA序列的反向互补序列：sgRNAl’：

sgRNA2’：

sgRNA3’：

在5’端添加AAAC，形成负链sgRNA DNA序列。In the three sgRNA guide sequences sgRNA1:

sgRNA2:

sgRNA3:

Add CACCG at its 5' end to form a positive-strand sgRNA DNA sequence; simultaneously synthesize the reverse complementary sequence of the positive-strand sgRNA DNA sequence: sgRNA1':

sgRNA2':

sgRNA3':

AAAC is added at the 5' end to form a negative-strand sgRNA DNA sequence.

The three pairs of sgRNA sequences are as follows:

sgRNA1F:

sgRNA1R:

sgRNA2F:

sgRNA2R:

sgRNA3F:

sgRNA3R:

2. Construction of targeting vector:

(1) Anneal the above positive and negative strand sgRNA sequences to form a double-stranded DNA sequence. The reaction system is as follows:

The above reaction system was centrifuged briefly and incubated at 95°C for 3 minutes, and cooled naturally for 20 minutes.

(2) Using BbsI enzyme to linearize the knockout vector pGK2.1, the reaction system is as follows:

After incubation at 37°C for 4 hours, the kit purified and recovered the linearized vector pGK2.1.

(3) Ligate the double-stranded DNA sequence formed by the above-mentioned annealing with the linearized vector pGK2.1, and the reaction system is as follows:

After the above system was centrifuged briefly, it was incubated at 16° C. for 30 min to obtain the ligation product.

(4) Add 10 μl of the above ligation product to DH5α competent cells, mix gently, and incubate on ice for 30 minutes; heat shock at 42°C for 60 sec, quickly take it out and cool it on ice for 2-3 minutes; add 800 μl of no-antibody SOC to the tube Liquid culture medium, cultivated on a shaking table (37°C/160rpm) for 45min; centrifuged at 4500rpm for 5min, discarded 600μl supernatant, resuspended the pellet in the remaining 200μl supernatant, and spread evenly on the culture containing kanamycin resistance Plates were cultured overnight at 37°C upside down.

Design three pairs of PCR primers based on the knockout vector pGK2.1 and three sgRNA sequences:

F1:

R1:

F2:

R2:

F3:

R3:

Colony PCR screening: Pick white monoclonal colonies from the above culture plate and place them in a PCR tube, add 1 μl of the above-mentioned upstream and downstream primers, 2 μl of buffer (10X), 2 μl of dNTP, and 0.2 μl of Taq enzyme into the PCR tube , 13.8 μl of double distilled water. The PCR reaction program was 95°C for 10 min; 35 cycles of 95°C for 35 sec, 60°C for 35 sec, and 72°C for 35 sec; 72°C for 10 min.

Figure 1 is a PCR gel electrophoresis detection map of colonies, where M is a 100bp DNA Marker, and 1, 2, and 3 are positive colony PCR bands. It can be seen from Figure 1 that the above method has obtained a positive targeting vector.

The obtained positive colony PCR products were further sequenced and verified, as shown in Figure 2, the positive targeting vector gene sequence was consistent with the pGK2.1 vector and sgRNA sequence. The positive targeting vectors were expanded and cultivated by conventional methods, and plasmids were extracted for subsequent experiments.

3. Cell transfection:

About 2× ¹⁰⁶ porcine intestinal epithelial cells (IPEC-J2) in the logarithmic growth phase were placed in a 15mL centrifuge tube, the supernatant was discarded by centrifugation at 1000rpm/5min, the cells were suspended with 210μl DPBS, and transferred to a 1.5mL centrifuge tube. Add 5-8 μg of the above three targeting carriers to the above-mentioned 1.5mL centrifuge tube containing the IPEC-J2 cell suspension, mix gently, transfer the mixture to the electric shock cup with a special electric transfer gun tip, and wait until the liquid surface rises. Finally, cover the electric shock cup and switch to the electroporation instrument at 650V/30ms for electroporation, and then transfer the cell solution to the culture medium of the six-well plate.

4. Knockout of KLF4 gene:

After 72 hours of electroporation, puromycin at a concentration of 3 μg/ml was used for drug screening, and the cells were diluted to 10 96-well plates by the limiting dilution method. After static culture in a _CO2 incubator at 37°C for 7 days, the monoclonal colonies were observed. In terms of growth, after about 14 days, the monoclonal cells were transferred to 48 wells for expanded culture. When the cells covered the 48 wells, some cells were isolated, and their genomic DNA was extracted using a DNA extraction kit.

5. PCR amplification of the DNA extracted in step 4:

According to the full sequence of the KLF4 gene, design highly specific primers near the knockout target site, the sequence is:

F:

R :

The PCR amplification reaction system is as follows:

The PCR reaction program was 95°C for 5 min; 95°C for 10 sec, 60°C for 10 sec, 72°C for 10 sec, 35 cycles; 72°C for 5 min, and 95°C for 3 min to obtain a PCR product with a length of 326 bp.

Fig. 3 is a gel electrophoresis detection diagram of PCR amplification of the knockout target site. Among them, M is a 100bp DNA Marker, and 1, 2, and 3 are PCR product bands. It can be seen from Figure 3 that the primers designed near the knockout target site can successfully amplify the target sequence.

6. Screening and verification of KLF4 knockout cells:

(1) Cruiser ^TM digestion and selection of KLF4 gene knockout cells

Use the Cruiser ^TM enzyme that recognizes mismatches to detect the above-mentioned PCR product with a length of 326 bp, and configure the reaction system as follows:

After reacting at 45°C for 20 min, 2 μl of 6×Stop Buffer was added to the above 10 μl reaction system, and then detected by agarose gel electrophoresis. Figure 4 shows the results of Cruiser ^TM digestion and screening of KLF4 gene knockout cells, where M represents 100bpDNAMarker; 1-1, 1-2 represent cells transfected with sgRNA1 targeting vector; 2-1, 2-2 represent cells transfected with sgRNA2 targeting vector Cells; 3-1, 3-2 represent cells transfected with sgRNA3 targeting vector. It can be seen from Figure 4 that sgRNA1 and sgRNA2 cannot effectively knock out the KLF4 gene, while sgRNA3 can effectively knock out the KLF4 gene. The targeting efficiency was analyzed according to the gray value, and the results showed that the KLF4 gene targeting efficiencies in the cells corresponding to 3-1 and 3-2 were 54.8% and 56.1%, respectively.

(2) DNA PCR product sequencing verification of KLF4 gene knockout cells:

The DNA PCR products of KLF4 gene knockout cells screened by Cruiser ^TM enzyme digestion were sequenced and verified, as shown in the sequencing peak diagram in Figure 5, it was determined that the cells transfected with the sgRNA3 targeting vector were KLF4 gene knockout cells. The DNA of the KLF4 gene knockout cells was cloned by TA and then sequenced, and compared with the DNA sequences of the KLF4 gene knockout cells, the DNA sequences of the knockout cells were mutated into deletions of 116bp and 137bp (Figure 6).

Figure 6 shows the KLF4 gene knockout sequence analyzed by TA clone sequencing. Among them, the first line is the sequence of KLF4 gene knockout, and the second and third lines are the KLF4 gene knockout sequence.

The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

sequence listing

<110> Yangzhou University

<120> A kind of sgRNA, knockout vector, KLF4 gene knockout method and its application

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<170> SIPOSequenceListing 1.0

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<212>DNA

<213> sgRNA1 (Artificial Sequence)

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acgttattcg gggcacctgc 20

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<212>DNA

<213> sgRNA2(Artificial Sequence)

<400> 2

acaacactca cgttattcg 19

<210> 3

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<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)

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cgaataacgt gagtgttgt 19

<210> 8

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<212>DNA

<213> F3(Artificial Sequence)

<400> 8

catatgctta ccgtaacttg aaag 24

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<212>DNA

<213> R3(Artificial Sequence)

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ggcgagtctg acatggctgt 20

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<212>DNA

<213> sgRNA1F(Artificial Sequence)

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caccgacgtt attcggggca cctgc 25

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<212>DNA

<213> sgRNA1R(Artificial Sequence)

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aaacgcaggt gccccgaata acgtc 25

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<212>DNA

<213> sgRNA2F(Artificial Sequence)

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caccgacaac actcacgtta ttcg 24

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<212>DNA

<213> sgRNA2R(Artificial Sequence)

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aaaccgaata acgtgagtgt tgt 23

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<212>DNA

<213> sgRNA3F(Artificial Sequence)

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caccgacagc catgtcagac tcgcc 25

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<212>DNA

<213> sgRNA3R(Artificial Sequence)

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aaacggcgag tctgacatgg ctgt 24

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<212>DNA

<213> F(Artificial Sequence)

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ggacttcgga ggacctctga 20

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<212>DNA

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ccaccccacag 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 ℃.

Images