CN110229816B - Construction method and application of sgRNA for knocking out RBP4 gene, RBP4 gene deletion cell line - Google Patents

Abstract

The present invention provides a sgRNA sequence for knocking out the RBP4 gene, the target DNA sequence of the sgRNA is at least one of the sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. The present invention also provides a method for knocking out the RBP4 gene of human liver cancer cells, which uses the CRISPR/Cas system to modify the RBP4 gene in human liver cancer cells. The present invention also provides a RBP4 gene-deficient cell line. RBP4 is a new type of fat factor, which has the function of interfering with glucose and lipid metabolism, and can also negatively regulate the insulin signaling pathway to cause insulin resistance. The RBP4 gene knockout cell line provided by the invention provides an effective platform for researching the RBP4 gene, and is expected to provide new targets for the treatment of diseases such as diabetes, metabolic syndrome, and obesity.

Description

Construction method for sgRNA for knocking out RBP4 gene, RBP4 gene deletion cell line and applications

technical field

The present invention relates to the field of genetic engineering, in particular to a sgRNA sequence for knocking out the RBP4 gene, a method for constructing a RBP4 gene-deficient cell line, and an application thereof.

Background technique

The CRISPR/Cas9 system is an acquired immune defense system to protect bacteria or archaea from the invasion of foreign plasmids or phages. The CRISPR sequence in the genome of such bacteria or archaea can express RNA that recognizes the genome sequence of the invader. Under the action of CRISPR-associated enzyme (CAS9), exogenous genomic DNA is cut to achieve the purpose of resisting invasion. After artificial modification, the CRISPR/Cas9 system can realize highly flexible and specific genome editing in eukaryotic cells. The most popular new-generation genome editing technology in the field, this technology has been used to construct various gene knockout cell lines and gene knockout animal models.

Among the existing experimental technologies, the most similar to the CRISPR/Cas9 system is the siRNA-targeted gene silencing technology. siRNA refers to the gene silencing phenomenon induced by highly conserved double-stranded RNA in the evolution process of organisms, which affects a series of processes and functions of organisms, but under the induction of drugs, etc., the expression of silenced genes will appear to recover. Compared with siRNA targeting mRNA to inhibit gene expression, the CRISPR/Cas9 system performs gene editing at the genome level, can completely silence gene expression, and construct a truly gene-deficient cell line.

Retinol-binding protein 4 (RBP4) is a specific carrier protein for retinol, vitamin A, and the like. RBP4 is a new type of adipokine, which has the function of interfering with glucose and lipid metabolism, and can also negatively regulate the insulin signaling pathway to cause insulin resistance. The RBP4 gene is located in the region of chromosome 10q23-q24, and most of the genes in this region are related to the occurrence and development of type 2 diabetes. Research on RBP4 gene is expected to provide new targets for the treatment of diabetes, metabolic syndrome, obesity and other diseases.

Therefore, it is necessary to develop a RBP4 gene-deficient cell line that can achieve complete silencing and long-term stable in vitro culture. It is expected that the study of RBP4 gene-deleted cell lines will provide new targets for the treatment of diabetes, metabolic syndrome, obesity and other diseases.

Contents of the invention

In view of this, the present invention provides the sgRNA sequence for knocking out the RBP4 gene, the construction method of the RBP4 gene deletion cell line and its application.

The first aspect of the present invention provides a sgRNA sequence for knocking out the RBP4 gene, the target DNA sequence of the sgRNA is at least one of the sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 .

The second aspect of the present invention provides a method for knocking out the RBP4 gene. In order to use the CRISPR/Cas system to modify the RBP4 gene in human liver cancer cells, it specifically includes the following steps:

1) Artificially synthesizing the target DNA sequence and its complementary strand as described in the first aspect of the present invention;

2) Insert the synthesized nucleic acid fragment into the multiple cloning site of the sgRNA backbone expression plasmid vector and transform it, pick a single clone strain, extract the sgRNA recombinant plasmid, sequence and identify the correct sgRNA recombinant plasmid; wherein, the sgRNA backbone expression plasmid vector Also expresses the Cas9 nuclease;

3) Transfecting the sgRNA recombinant plasmid into human liver cancer cells to obtain human liver cancer cells in which the RBP4 gene has been knocked out.

In an embodiment of the present invention, the step 2) specifically includes: inserting the synthesized nucleic acid pairs of sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 into the sgRNA backbone expression plasmid vector Multiple cloning sites and transformation, single-clonal strains were picked, sgRNA recombinant plasmids were extracted, sequenced and identified to obtain the correct sgRNA recombinant plasmids; wherein, the sgRNA backbone expression plasmid vector also expressed Cas9 nuclease;

In an embodiment of the present invention, the step 3) specifically includes: co-transfecting human liver cancer cells with the sgRNA recombinant plasmid obtained in step 2), to obtain human liver cancer cells in which the RBP4 gene is knocked out.

In the third aspect, the present invention provides a method for constructing a RBP4 gene-deficient cell line, which is to use the lentiviral transfection method to perform subculture screening on the RBP4 gene-knockout human liver cancer cells obtained in the second aspect of the present invention, and obtain a stable knockout of the RBP4 gene human liver cancer cells.

In the fourth aspect, the present invention provides a RBP4 gene-deleted cell line, which is obtained by adopting the method for constructing the RBP4 gene-deleted cell line described in the third aspect of the present invention.

In the fifth aspect, the present invention provides a sgRNA sequence for knocking out the RBP4 gene as described in the first aspect, the method for knocking out the RBP4 gene as described in the second aspect, and the RBP4 gene as described in the third aspect of the present invention. A method for constructing a gene-deleted cell line or an application of the RBP4 gene-deleted cell line according to the fourth aspect of the present invention in knocking out the RBP4 gene.

In the sixth aspect, the present invention provides a kit for performing targeted knockout of RBP4 gene in human liver cancer cell genome in vitro, including any one of the following 1)-3):

1) the sgRNA sequence for knocking out the RBP4 gene as described in the first aspect;

2) sgRNA recombinant plasmid as described in the second aspect;

3) The RBP4 gene deletion cell line as described in the fourth aspect.

The technical scheme provided by the invention has the following beneficial effects:

The technical solution provided by the present invention utilizes CRISPR/Cas9 technology to knock out the RBP4 gene, realizes gene silencing at the genome level, and effectively improves the shortcomings of incomplete silencing of siRNA at the mRNA or protein level or inability to silence gene expression.

Description of drawings

Fig. 1 is the PCR fragment result after the SURVEYOR nuclease digestion that the embodiment of the present invention provides;

Fig. 2 is the T-A clone sequencing result of the RBP4 gene knockout HepG2 stable strain provided by the embodiment of the present invention;

Fig. 3 is the detection result of the mRNA expression level of the HepG2 cell line of RBP4 gene knockout provided by the embodiment of the present invention;

Fig. 4 is the detection result of Protein expression in the RBP4 gene knockout HepG2 cell line provided by the embodiment of the present invention.

Detailed ways

The following description is a preferred embodiment of the present invention, it should be pointed out that for those skilled in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications are also considered Be the protection scope of the present invention.

Unless otherwise specified in the examples of the present invention, all reagents and consumables used are commercially available.

(1) sgRNA design:

In this study, the http://crispr.mit.edu/ website was used to design the sgRNA sequence for the third exon (Exon3) of RBP4.

Design method:

1) Search the sequence of the third exon (Exon3) of the RBP4 gene on NCBI.

2) Submit the Exon3 sequence released by NCBI to the above website. After submission, the website will design the sgRNA by itself. According to the results of the website design, select a pair of sequences with a higher score (generally, the selection score is >80).

3) Add restriction sites at both ends of the sgRNA, add CACC to the 5' end of the sense strand of each sgRNA sequence, and add AAAC to the 5' end of the antisense strand, thereby forming a fusion with the pLentiCRISPRv.2 plasmid through Fast Digest BsmbI enzyme Complementary cohesive ends after cleavage. If the first base at the 5' end of the sense strand is not G, add a G after CACC at the 5' end, and add a C at the 3' end of the corresponding antisense strand. After the design is completed, the sgRNA with enzyme cutting sites will be sent to Shanghai Jierui Bioengineering Co., Ltd. for synthesis. The specific grouping and naming of the three groups of sgRNA sequences with restriction sites for Exon3 of RBP4 are shown in the following table:

Table 1 RBP4 sgRNA oligo sequences

The sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 of the present invention correspond to the horizontal lines of SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8 in Table 1, specifically , the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 are GAGTTCTCCGTGGACGAGAC, CTTCTTGGCCATGGCGTACC, CATCGTCGCGGAGTTCTCCG, respectively.

(2) Construction and identification of recombinant plasmids

1) The pLentiCRISPRv.2 (addgene company, product number 52961) plasmid is an sgRNA backbone expression vector containing a U6 promoter, expressing a Cas9 nuclease capable of cleaving DNA double strands to generate DNA double strand breaks (DNA Double Strand Break, DSB), with Ampicillin resistance and puromycin resistance. pLentiCRISPRv.2 was digested with Fast Digest BsmbI, and the linearized vector was recovered after DNA gel electrophoresis.

2) Use T4 PNK to phosphorylate and anneal the sgRNA oligo sequences in Table 1 respectively; use T4ligase to ligate the linear pLentiCRISPRv.2 plasmid vector and the three sets of annealed sgRNA double-stranded sequences at 16°C for 2 hours. The ligation product was transformed into competent bacteria Trans109, kept in an ice bath for 30 minutes, then heat-stressed in a water bath at 42°C for 45 seconds, and kept on ice for 2 minutes. Clones were selected on LB plates containing ampicillin resistance. Pick positive clones and send them to Beijing Liuhe Huada Gene Company for sequencing. Correctly sequenced clones were used to extract recombinant plasmids.

3) There are three groups of recombinant plasmids obtained, and the recombinant plasmids obtained for the pLentiCRISPRv.2 plasmid are named pLentiCRISPRv.2-sgRNA1, pLentiCRISPRv.2-sgRNA2 and pLentiCRISPRv.2-sgRNA3.

(3) Construction of HepG2 stable cell line with virus liquid of lentivirus system

1) Virus production:

The culture conditions of 293FT cells are DMEM medium (containing 10% fetal bovine serum), 5% CO ₂ , and constant temperature culture at 37°C.

293FT cells were inoculated in 10 cm cell culture dishes respectively, and the inoculated number of cells was 2×10 ⁶ to ensure that the cell fusion degree reached about 60% the next day. HEK293FT cells were co-transfected with core plasmid (pLentiCRISPRv.2-sgRNA or pLentiCRISPRv.2), packaging plasmid psPAX2, and envelope plasmid pMD2.G at a ratio of 4:3:2. The core plasmids of the sgRNA group were pLentiCRISPRv.2-sgRNA1, pLentiCRISPRv.2-sgRNA2 and pLentiCRISPRv.2-sgRNA3, and pLentiCRISPRv.2 was used as the control group. At 24h, 48h and 72h after transfection, the virus liquid was collected three times continuously, and the virus liquid collected each time was filtered with a 0.45 μm filter membrane to remove cell debris and other impurities. Determine the transfection efficiency by observing the percentage of fluorescent cells using an inverted fluorescence microscope.

2) Cell infection and screening of HepG2 stable strain cells:

The HepG2 cell culture conditions are DMEM medium (containing 10% fetal bovine serum), 5% CO ₂ , and constant temperature culture at 37°C.

One day in advance, HepG2 cells were seeded in 6-well plates at 2×10 ⁵ /well, and the cell confluency should reach 30% when the cells were infected. Before infection, mix the virus stock solution with an equal volume of fresh medium, add 2 μl of 10 mg/ml polybrene (final concentration is 10 μg/ml) and mix well, then add it into a six-well plate. So repeatedly infected three times. After infection, after the cell confluency reaches 80-90%, the infected cells are screened with puromycin at a final concentration of 1 μg/ml. After 5 days of drug screening, 0.2 μg/ml puromycin was used to maintain the stable HepG2 cell line.

(4) Extraction of cellular genomic DNA

The constructed cell line was digested, and the genomic DNA was extracted with GeneJET ^TM Genomic DNA Purification Kit.

1) Collect the cells in centrifuge tubes, 5×10 ⁶ cells per tube, blow slowly with a pipette, centrifuge at 250g for 5 minutes, discard the supernatant, add PBS to resuspend the cells, and repeat the centrifugation again to remove the residual medium in the cells .

2) Resuspend the cells in 200 μl PBS, add 200 μl lysate and 20 μl proteinase K to each tube, shake well and mix well.

3) Incubate on a shaker at 56°C for 10 minutes, and shake and mix every 3-4 minutes during the period to ensure that the cells are fully lysed.

4) Add 20 μl RNAase A, shake and mix, and incubate at room temperature for 10 minutes.

5) Add 400 μl of 50% ethanol and mix with a gun or oscillate.

6) Add the above-mentioned MiX to the Column column provided in the kit, centrifuge at 6000g for 1 min, and transfer the DNA collection column to a new 2ml collection tube.

7) Add 500μl wash buffer I, centrifuge at 8000g for 1min, and discard the waste liquid. Then add 500μl washbuffer II, centrifuge at 12000g for 3min.

8) Add 200 μl Elution Buffer to the center of the filter membrane of the collection column, incubate at room temperature for 2 minutes, centrifuge at 8000 g for 1 minute, and obtain the desired DNA sample.

(5) PCR reaction conditions and SURVEYOR analysis and detection

1) Design QRT-PCR primers according to the RBP4 gene mRNA sequence number provided by NCBI; design SURVEYOR primers and PCR primers for the RBP4 gene through Primer 3.0, and the primers were synthesized by Shanghai Jierui Biotechnology Company.

Table 2 SURVEYOR PCR reaction primer sequence

2) Perform PCR amplification with Phusion ultra-fidelity DNA polymerase, refer to the manual for 50 μl system, genomic DNA 100ng, 50 μl reaction system is shown in the table below.

components quantity 5×Phusion HF buffer 10μl dNTPs, 2.5mM 4μl Forward primer (F+R, 10uM) 2..5μl template DNA 100ng ddH2O Up to 50μl

The procedure is as follows:

After the reaction, 5 μl of the PCR product was taken for agarose gel electrophoresis to detect its specificity.

The SURVEYOR analysis steps are as follows:

1) Purify the PCR product with the QIAquick PCR purification Kit kit, dilute the recovered product to 40ng/ul, and detect it according to the instructions of the SURVEYOR analysis kit.

2) DNA hybrid double strand formation (annealing reaction) system:

components Amount (μl) 10×Taq PCR buffer 2 PCR product after purification, 50ngμl ^-1 18 Total volume 20

Reaction conditions:

Cycles condition 1 95℃,10min 2 95-85℃, -2℃s ^-1 3 85℃,1min 4 85-75℃,-0.3℃s ^-1 5 75℃,1min 6 75-65℃,-0.3℃s ^-1 7 65℃,1min 8 65-55℃,-0.3℃s ^-1 9 55℃, 1min 10 55-45℃,-0.3℃s ^-1 11 45℃,1min 12 45-35℃,-0.3℃s ^-1 13 35℃,1min 14 35-25℃,-0.3℃s ^-1 15 25℃,1min 16 25-4℃,-0.3℃s ^-1 17 4℃, hold

3) Use the nuclease provided by the SURVEYOR kit to digest the hybrid product, the system is as follows:

Reaction conditions: fully oscillate and mix the above mixture, 42°C for 30min.

f _cut＝(b+c)/(a+b+c)，其中Indel为缺失比率，f_cut为切割比率，a为未被切割条带的灰度值，b和c表示切割产生的新条带的灰度值。4) Take 10ul sample and analyze it with 2.5% agarose gel. Use the gel quantification software Image Lab to carry out grayscale analysis, calculate the cutting efficiency, the formula is

f _cut = (b+c)/(a+b+c), where Indel is the missing ratio, f _cut is the cutting ratio, a is the gray value of the uncut strip, b and c represent the new strip produced by cutting The gray value of the band.

Select the group of transfected 293FT cells with the highest Indel (%) for next inoculation and screening.

The results of the SURVEYOR nuclease digestion part are shown in Figure 1. The experimental results showed that the pLentiCRISPRv.2-sgRNA1, pLentiCRISPRv.2-sgRNA2 and pLentiCRISPRv.2-sgRNA3 plasmid groups constructed for the sgRNA designed for Exon3 were all effective, and the SURVEYOR results were positive. Among them, the Indel (%) was the highest after being treated with the pLentiCRISPRv.2-sgRNA3 plasmid group, so the 293FT cells transfected with the pLentiCRISPRv.2-sgRNA3 plasmid group were selected for the next step of inoculation and screening.

(6) DNA sequencing analysis of RBP4 gene knockout HepG2 cell line

1) Use NEB company Taq enzyme to perform PCR amplification on the third exon region of RBP4, the reaction system:

2) Ligate the PCR product into the T vector provided by the kit, the ligation system is as follows:

Reaction conditions: react at room temperature (22-30°C) for 1-5 minutes.

3) Introduce the connection solution into the competent cells, heat shock in a 42°C water bath for 45 seconds after ice bath for 30 minutes, quickly transfer back to the ice bath, and let stand for 2 minutes; add 500 μl of sterile LB medium without antibiotics, and It was placed at 37° C. and shaken on a shaker at 300 rpm for one hour. Pipette 500 μl of bacterial liquid and evenly spread it on the LB culture plate containing antibiotics;; Invert the plate, place it at 37°C for overnight culture, pick a single clone and send it for sequencing the next day.

The results are shown in Figure 2a, Figure 2b, and Figure 2c, the HepG2 stable strain caused a deletion mutation at the target site, and knocked out the RBP4 gene.

(8) Detection of mRNA and protein expression of RBP4 gene knockout HepG2 cell line

1) Total cellular RNA was extracted using the Trizol method, reverse-transcribed using a TOYOBO reverse transcription kit to generate cDNA, and qRT-PCR was performed according to the system given in the TOYOBO SYBR mix reagent manual.

Design qRT-PCR primers according to the RBP4 gene mRNA sequence number provided by NCBI;

The cDNA was obtained by reverse transcription using the TOYOBO reverse transcription kit. The system is as follows:

Perform real-time fluorescent quantitative PCR according to the system on TOYOBO's SYBR Mix kit manual:

Real-time fluorescent quantitative PCRVII7 instrument PCR reaction cycle conditions are as follows:

The results are shown in Figure 3, the mRNA expression level of the HepG2 cell line with successfully constructed RBP4 gene knockout was significantly lower than that of the control group.

2) The total protein of HepG2 RBP4 gene knockout cell line was extracted using M-PER cell lysate, and quantified using the BCA protein quantification kit of Shanghai Beyontien Biotechnology Co., Ltd., followed by Western Blotting.

Configure X% separation gel (8mL):

Fix it with a Bio-Rad gel frame, seal the liquid surface with absolute ethanol on the upper layer, and place it at 37°C for about 30 minutes until the gel is completely solidified.

Prepare 4% stacking gel (6mL):

Insert the sample comb after filling the glue, and place it at 37°C for about 30 minutes until the glue is completely solidified.

Sample processing and sample loading: Calculate the protein volume based on the protein amount of 30 μg/well, add 10 μl of 5×SDS buffer, and make up the total volume to 20 μl with protein lysate. Denature at 98°C for 8 minutes, centrifuge at 12,000rpm for 1 minute, then slowly add to the sample well.

The results are shown in Figure 4, the protein expression of the HepG2 cell line successfully constructed to knock out the RBP4 gene was significantly lower than that of the control group.

The above results show that the method for constructing RBP4 gene-deleted cell lines provided by the present invention is an efficient gene knockout method. The traditional gene knockout method is not only cumbersome, requires high technical requirements, but also is expensive and has a relatively low success rate. The CRISPR-Cas9 technology adopted in the present invention is a fourth-generation gene editing method, which is easy to operate, has higher efficiency and is low in cost. The RBP4 gene knockout cell model constructed by using CRISPR-Cas9 technology in the present invention provides an effective platform for research related to RBP4 metabolism.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. within range.

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Claims (6)

1. A sgRNA sequence for knocking out the RBP4 gene, characterized in that the target DNA sequence of the sgRNA is as shown in SEQ ID NO:3. 2. A non-disease diagnosis and treatment method for knocking out the RBP4 gene of human liver cancer cells, characterized in that, for utilizing the CRISPR/Cas system to transform the RBP4 gene in human liver cancer cells in vitro, specifically comprising the following steps: 1) artificially synthesizing the target DNA sequence and its complementary strand as claimed in claim 1; 2) Insert the synthesized nucleic acid fragment into the multiple cloning site of the sgRNA backbone expression plasmid vector and transform it, pick a single clone strain, extract the sgRNA recombinant plasmid, and sequence and identify the correct sgRNA recombinant plasmid; wherein, the sgRNA backbone expression plasmid vector Also expresses the Cas9 nuclease; 3) The sgRNA recombinant plasmid was transfected into human liver cancer cells by lentiviral transfection method to obtain human liver cancer cells with RBP4 gene knockout. 3. A method for constructing a RBP4 gene-deleted cell line, characterized in that, the human liver cancer cells with RBP4 gene knockout as claimed in claim 2 are subcultured and screened to obtain stable RBP4-knockout human liver cancer cells. 4. A RBP4 gene-deficient cell line, characterized in that it is prepared by the construction method of RBP4 gene-deleted cell line as claimed in claim 3. 5. The application of the sgRNA sequence for knocking out the RBP4 gene as claimed in claim 1 in the preparation of a kit for targeted knockout of the RBP4 gene in the genome of human liver cancer cells in vitro. 6. A kit for performing site-directed knockout of RBP4 gene in human liver cancer cell genome in vitro, characterized in that, comprising any one of the following 1)-2): 1) the sgRNA sequence for knocking out the RBP4 gene as claimed in claim 1; 2) sgRNA recombinant plasmid as claimed in claim 2.

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