CN113801880B - Early senescence cell model established by targeted knockout gene, sgRNA (single-stranded ribonucleic acid) thereof, construction method and application - Google Patents

Abstract

The present disclosure provides a premature aging cell model established by targeted gene knockout, and its sgRNA, construction method and application. The double sgRNA targeted knockout method is adopted to simultaneously generate two DNA double-strand break points in the second exon of the GDF11 gene and the intron sequence upstream thereof, and the two sites on the second exon of the GDF11 gene are cut, with high knockout efficiency. At the same time, the identification means are simple and the identification efficiency is high. Only PCR amplification and electrophoresis are required to determine whether the knockout is successful, which greatly reduces the identification cost after knockout. A neuroblastoma cell line, namely the Neuro‑2a cell line, in which the GDF11 gene is knocked out is established, which provides an in vitro cell model for exploring the biological function of endogenous GDF11. Based on the experimental results, it is found for the first time that the Neuro‑2a cell line in which the GDF11 gene is knocked out has aging characteristics, which provides a research tool for research in the field of aging, promotes research on anti-aging topics, and provides a research basis for delaying aging.

Description

A premature aging cell model established by targeted gene knockout and its sgRNA, construction method and application

Technical Field

The present disclosure relates to the field of genetic engineering technology, and in particular to a progeria cell model established by targeted gene knockout, and its sgRNA, construction method and application.

Background Art

Cellular senescence is a phenomenon in which cells undergo irreversible cell cycle arrest, macromolecular damage, secretory phenotype formation, and metabolic disorders, involving multiple characteristics of different physiological and age-related diseases. Most studies on cellular senescence focus on highly proliferative cells, as cell proliferation causes telomere shortening, which in turn causes cell replicative senescence.

Recent studies on the aging of organs such as the brain and retina, which are mainly composed of terminally differentiated neurons, have shown that terminally differentiated cells can also undergo cellular senescence, and their aging mechanism is different from that of highly proliferative cells. The specific mechanism has just been revealed, and there is still a lack of cellular models of premature aging.

Summary of the invention

In view of this, the purpose of the present disclosure is to propose a premature aging cell model established by targeted gene knockout and its sgRNA, construction method and application, so as to provide a basis for studying the aging mechanism of terminally differentiated cells.

Based on the above purpose, the first aspect of the present disclosure provides a dual sgRNA for targeted knockout of the GDF11 gene, including GDF11-sgRNA1 and GDF11-sgRNA2;

The targeting sequence of the GDF11-sgRNA1 is: GCCGAAGGTACACCCACAGT, (SEQ ID NO: 1)

The targeting sequence of the GDF11-sgRNA2 is: GAACCGGGTAAGGTAGCTTG. (SEQ ID NO: 2)

Optionally, the nucleotide sequence of the GDF11-sgRNA1 includes:

GDF11-sgRNA1 Oligo1: 5'—CACCGGCCGAAGGTACACCCACAGT—3', (SEQ ID NO: 3)

GDF11-sgRNA1 Oligo2: 5'-AAACACTGTGGGTGTACCTTCGGCC-3'; (SEQ ID NO: 4)

The nucleotide sequence of the GDF11-sgRNA2 includes:

GDF11-sgRNA2 Oligo1: 5'—CACCGGAACCGGGTAAGGTAGCTTG—3', (SEQ ID NO: 5)

GDF11-sgRNA2 Oligo2: 5'—AAACCAAGCTACCTTACCCGGTTCC—3'. (SEQ ID NO: 6)

The present invention adopts a dual sgRNA targeted knockout method to simultaneously generate two DNA double-strand break points in the second exon of the GDF11 gene and its upstream intron sequence, and cuts the two sites on the second exon of the GDF11 gene, with high knockout efficiency. At the same time, the identification method is simple and the identification efficiency is high. Only PCR amplification and electrophoresis are needed to determine whether the knockout is successful, which greatly reduces the identification cost after knockout.

Furthermore, the present disclosure also provides a DNA molecule for encoding the dual sgRNA for targeted knockout of the GDF11 gene as described in the first aspect of the present disclosure.

Based on the same purpose, the second aspect of the present disclosure provides a method for constructing a Neuro-2a cell line for targeted knockout of the GDF11 gene, comprising knocking out the GDF11 gene of the Neuro-2a cell line using the CRISPR/Cas9 system, wherein the dual sgRNA used in the CRISPR/Cas9 system is the dual sgRNA described in the first aspect of the present disclosure.

Optionally, the method for constructing Neuro-2a cell line with targeted knockout of GDF11 gene comprises:

Step 1, designing the targeting sequence of the dual sgRNA according to the GDF11 gene, and obtaining the nucleotide sequences of the dual sgRNA respectively;

Step 2, connect the nucleotide sequences of the dual sgRNAs to the pX459 vector respectively to obtain the expression vector of the dual sgRNAs;

Step 3, culturing the Neuro-2a cell line, and co-transfecting the expression vectors of the dual sgRNAs into the Neuro-2a cell line;

Step 4, screening, enriching, sorting and culturing to obtain puromycin-resistant Neuro-2a positive cells;

Step 5, extracting the genome of the puromycin-resistant Neuro-2a positive cell line and performing PCR amplification;

Step 6: Sequence the PCR amplification product and compare the sequencing result with the normal uncut reference sequence SEQ ID NO: 9 to determine whether it is correctly cut.

Optionally, the pX459 vector is used in knocking out the GDF11 gene of the Neuro-2a cell line using the CRISPR/Cas9 system.

Optionally, the dual sgRNAs are ligated to the vector pX459 by:

The sequences SEQ ID NO: 3 and SEQ ID NO: 4 were connected to the first pX459 vector after BbsI digestion to construct the expression vector pX459-U6-1-sgRNA-CMV-Cas9-T2A-PuroR; the sequences SEQ ID NO: 5 and SEQ ID NO: 6 were connected to the second pX459 vector after BbsI digestion to construct the expression vector pX459-U6-2-sgRNA-CMV-Cas9-T2A-PuroR.

Based on the same purpose, the third aspect of the present disclosure provides a Neuro-2a cell line with GDF11 gene knockout obtained according to the method for constructing a Neuro-2a cell line with GDF11 gene knockout according to the second aspect of the present disclosure.

Furthermore, the present disclosure also provides a primer pair for identifying the mutation type of the Neuro-2a cell line in which the GDF11 gene is knocked out, the primer pair comprising:

The nucleotide sequences of the primers are shown in SEQ ID NO:7 and SEQ ID NO:8.

Based on the same purpose, the fourth aspect of the present disclosure provides a use of the Neuro-2a cell line with GDF11 gene knockout according to the third aspect of the present disclosure in preparing a GDF11 gene function research model.

Furthermore, the present disclosure also provides the use of Neuro-2a cell line with GDF11 gene knockout in preparing a disease model of cell senescence.

Furthermore, the present disclosure also provides the use of Neuro-2a cell line with GDF11 gene knockout in preparing a disease model of neuronal cell aging.

Furthermore, the present disclosure also provides the use of Neuro-2a cell line with GDF11 gene knocked out in screening drugs for treating or preventing cell senescence.

As can be seen from the above, the premature aging cell model established by targeted gene knockout provided by the present disclosure, and its sgRNA, construction method and application, adopt the dual sgRNA targeted knockout method, and simultaneously generate two DNA double-strand break points in the second exon of the GDF11 gene and its upstream intron sequence, and cut the two sites on the second exon of the GDF11 gene, with high knockout efficiency. At the same time, the identification method is simple and the identification efficiency is high. Only PCR amplification and electrophoresis are required to determine whether the knockout is successful, which greatly reduces the identification cost after knockout. A neuroblastoma cell line with GDF11 gene knockout, namely Neuro-2a cell line, is established, which provides an in vitro cell model for exploring the biological function of endogenous GDF11. Based on the experimental results, it is found for the first time that the Neuro-2a cell line with GDF11 gene knockout has aging characteristics, which provides a research tool for research in the field of aging, promotes the research of anti-aging topics, and provides a research basis for delaying aging.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions in the present disclosure or related technologies, the drawings required for use in the embodiments or related technical descriptions are briefly introduced below. Obviously, the drawings described below are only embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

FIG1 is a schematic diagram of the structure of the pX459 vector backbone BbsI digestion used in the embodiments of the present disclosure;

FIG2 is a schematic diagram of the sequencing results of the expression vector of GDF11-sgRNA1 in the embodiment of the present disclosure;

FIG3 is a schematic diagram of the sequencing results of the expression vector of GDF11-sgRNA2 in the embodiment of the present disclosure;

FIG4 is a schematic diagram of the PCR amplification results of the Neuro-2a cell line with GDF11 gene knocked out provided in an embodiment of the present disclosure;

FIG5 is a schematic diagram of the immunofluorescence staining results of the Neuro-2a cell line with GDF11 gene knocked out provided in an embodiment of the present disclosure;

FIG6 is a schematic diagram of the results of DAPI staining of the Neuro-2a cell line with GDF11 gene knockout provided in the embodiments of the present disclosure;

FIG. 7 is a schematic diagram of the results of aging-related β-galactosidase staining of the Neuro-2a cell line with GDF11 gene knockout provided in the embodiments of the present disclosure;

FIG8 is a schematic diagram of transmission electron microscopy results and statistical results of the Neuro-2a cell line with GDF11 gene knockout provided in an embodiment of the present disclosure.

DETAILED DESCRIPTION

In order to make the objectives, technical solutions and advantages of the present disclosure more clearly understood, the present disclosure is further described in detail below in combination with specific embodiments and with reference to the accompanying drawings.

It should be noted that, unless otherwise defined, the technical terms or scientific terms used in the embodiments of the present disclosure should have the usual meanings understood by people with ordinary skills in the field to which the present disclosure belongs. The words "first", "second" and similar words used in the embodiments of the present disclosure do not indicate any order, quantity or importance, but are only used to distinguish different components.

Cellular senescence is a phenomenon in which cells undergo irreversible cell cycle arrest, macromolecular damage, secretory phenotype formation, and metabolic disorders, involving multiple characteristics of different physiological and age-related diseases. Most studies on cellular senescence focus on highly proliferative cells, as cell proliferation causes telomere shortening, which in turn causes cell replicative senescence.

Recent studies on the aging of organs such as the brain and retina, which are mainly composed of terminally differentiated neurons, have shown that terminally differentiated cells can also undergo cellular senescence, and their aging mechanism is different from that of highly proliferative cells. The specific mechanism has just been revealed, and there is still a lack of cellular models of premature aging.

The GDF11 gene is one of the new members of the transforming growth factor β-superfamily (TGFβ). It participates in regulating the embryonic development of multiple organs and plays a vital role in early embryonic development. Experimental studies have found that through heterogeneous parabiosis surgery, the blood vessels of young and old mice are fused, so that they share the same blood circulation system. Previous research results have found that the aging characteristics of old mice, such as muscle atrophy, myocardial hypertrophy and neurological decline caused by aging, can be improved by young blood, and even reach the level of young people. The researchers then used protein screening technology to find that the GDF11 gene plays a key role in the above-mentioned reversal of aging. Therefore, the GDF11 gene was once considered to be a "rejuvenation" protein, which is closely related to aging and many related diseases. However, further studies have shown that in vitro recombinant rGDF11 gene inhibits the proliferation of stem cells and muscle regeneration in old mice, and has no effect on improving myocardial hypertrophy in old mice, and the opposite experimental results are obtained. Therefore, there is still a lot of controversy about the function of GDF11 gene in regeneration and delaying aging.

In order to explore whether the endogenous GDF11 gene plays a role in the aging of postmitotic neurons, the present invention uses the CRISPR/Cas9 system to specifically knock out the GDF11 gene in the Neuro-2a cell line, and uses monoclonal cell culture technology to construct the Neuro-2a cell line with GDF11 knocked out, so as to study the role of the GDF11 gene in anti-aging. This is described in detail in conjunction with the following specific examples.

Example 1. Construction and detection of CRISPR/Cas9 vector targeting GDF11 gene

1. Sequence design of GDF11 gene sgRNA

The sgRNA sequence was designed for the second exon of the mouse-derived GDF11 gene and its upstream intron sequence. The sgRNA was designed through the Chopchop website (https://chopchop.cbu.uib.no/). The GDF11 gene was knocked out by double-knife cutting, and two sgRNAs were designed, namely GDF11-sgRNA1 located in the second exon of the GDF11 gene and GDF11-sgRNA2 located in the upstream intron; the targeting sequence of GDF11-sgRNA1 was: GCCGAAGGTACACCCACAGT (as shown in SEQ ID NO: 1); the targeting sequence of GDF11-sgRNA2 was: GAACCGGGTAAGGTAGCTTG (as shown in SEQ ID NO: 2).

Restriction sites were designed at both ends of the sgRNA according to the BbsI restriction endonuclease, and CACCG was added to the 5' end of the sgRNA; AAAC was added to the 5' end of the reverse complementary sequence, and C was added to the 3' end. The nucleotide sequence of GDF11-sgRNA1 was obtained as follows:

GDF11-sgRNA1 Oligo1: 5'—CACCGGCCGAAGGTACACCCACAGT—3', (SEQ ID NO: 3)

GDF11-sgRNA1 Oligo2: 5'-AAACACTGTGGGTGTACCTTCGGCC-3'; (SEQ ID NO: 4)

The nucleotide sequence of GDF11-sgRNA2 includes:

GDF11-sgRNA2 Oligo1: 5'—CACCGGAACCGGGTAAGGTAGCTTG—3', (SEQ ID NO: 5)

GDF11-sgRNA2 Oligo2: 5'—AAACCAAGCTACCTTACCCGGTTCC—3'. (SEQ ID NO: 6)

It can be understood that the dual sgRNA targeted knockout method simultaneously generates two DNA double-strand break points in the second exon of the GDF11 gene and its upstream intron sequence, and cuts the two sites on the second exon of the GDF11 gene, with high knockout efficiency. At the same time, the identification method is simple and efficient. Only PCR amplification and electrophoresis are required to determine whether the knockout is successful, which greatly reduces the identification cost after knockout.

2. Construction of pX459 expression vector

(1) The pX459 vector has two BbsI restriction sites. After BbsI restriction, sticky ends are generated as shown in Figure 1. The restriction system is prepared according to Table 1. After preparation, the system is placed in a 37°C water bath for 4 to 5 hours. After the restriction is completed, 1% agarose gel electrophoresis is performed, and the restriction products containing sticky ends are recovered and purified using the Tiangen gel recovery kit.

Table 1. Preparation of pX459 enzyme digestion system reagents

(2) sgRNA Oligo annealing and double-strand formation. Prepare the oligo annealing system according to the dosage in Table 2 and place it in a PCR instrument. Heat the PCR instrument at 98°C for 2 minutes, turn off the PCR instrument immediately, and take out the reaction product after 1 hour; the GDF11-sgRNA single-stranded primer will be synthesized through the base pairing principle as the temperature slowly decreases to obtain a double-stranded GDF11-sgRNA containing a BbsI-cut sticky end.

Table 2. sgRNA double-stranded synthesis system

(3) The pX459 vector digested with BbsI was connected to the double-stranded GDF11-sgRNA containing the BbsI-digested sticky ends by T4 ligase, that is, the sequences SEQ ID NO: 3 and SEQ ID NO: 4 were connected to the first pX459 vector digested with BbsI to construct an expression vector; SEQ ID NO: 5 and SEQ ID NO: 6 were connected to the second pX459 vector digested with BbsI to construct an expression vector; the recombinant plasmids were screened, amplified and sequenced, and the sequencing results are shown in Figures 2 and 3, respectively.

As shown in Figures 2 and 3, the sequencing was correct, and the CRISPR/Cas9 expression vectors of the mouse GDF11 gene, namely pX459-U6-1-sgRNA-CMV-Cas9-T2A-PuroR and pX459-U6-2-sgRNA-CMV-Cas9-T2A-PuroR, were obtained.

Example 2: Construction and genome identification of Neuro-2a cell line with GDF11 gene knockout

1. Neuro-2a cell culture

Neuro-2a cells were cultured in a high-glucose DMEM medium containing 10% fetal bovine serum under 5% CO ₂ saturated humidity conditions. When the cell confluence reached about 90%, the medium was discarded, and the cells were washed once with 1x PBS (0.01 mol/L) and then digested with 0.25% trypsin for subculture.

2. Neuro-2a cell transfection

Neuro-2a cells were seeded into 24-well plates at a density of 0.5-1x10 ⁵ per well. When the cell density reached about 70%, lipo2000 liposomes were used for transfection. The transfection system is shown in Table 3.

Table 3. GDF11-sgRNA transfection system

3. CRISPR/Cas9 cleavage detection

48 hours after transfection, cells were collected and the genome of Neuro-2a cell line was extracted. According to the cleavage sites of GDF11-sgRNA1 and GDF11-sgRNA2, detection primers Check-exon2-F and Check-exon2-R were designed, and the nucleotide sequences of Check-exon2-F and Check-exon2-R were shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively; then PCR amplification was performed, the PCR reaction system was shown in Table 4, and the PCR reaction conditions were shown in Table 5. The length of the normal uncut PCR product was 992 bp, and the length of the PCR product after simultaneous cleavage of the target sites of GDF11-sgRNA1 and GDF11-sgRNA2 was about 514 bp.

Table 4. PCR reaction system

Table 5. PCR reaction conditions

4. Screening and enrichment of puromycin-resistant Neuro-2a positive cells

Neuro-2a cells were screened 48 hours after transfection using DMEM medium containing 3 μg/mL puromycin. DMEM medium containing 3 μg/mL puromycin was replaced every day for 4 consecutive days, and then DMEM medium without puromycin was used to collect the cells when the growth density of Neuro-2a cells reached 90%.

5. Isolation of Puromycin-resistant Neuro-2a positive cells

Collect the Neuro-2a positive cells 200g of resistance to puromycin, centrifuge for 5min, add 1mL DMEM culture medium to resuspend, and count with a cell counting plate. According to the cell concentration after counting, the culture medium containing about 100 Neuro-2a cells is drawn and added to 10mL DMEM culture medium, so that the final concentration is about: 1 cell/100μl, and the cells are planted in 96-well plates, 100μl per well. After 7 days of planting, observe under a microscope, screen out the cell line propagated by the single cell of Neuro-2a resistance to puromycin, collect cells when the cell growth density reaches 90%, plant them in 24-well plates, and continue to culture and proliferate to obtain monoclonal Neuro-2a positive cells of resistance to puromycin.

6. Identification of Neuro-2a positive cells

The Neuro-2a positive cells with GDF11 gene knocked out were compared with normal uncut Neuro-2a cells, and the nucleotide sequence of normal uncut Neuro-2a cells is shown in SEQ ID NO:9.

(1) Some monoclonal puromycin-resistant Neuro-2a positive cells were collected, the genome of the Neuro-2a cell line was extracted, and PCR amplification was performed using the detection primers Check-exon2-F and Check-exon2-R. The PCR reaction system was shown in Table 4 above, and the PCR reaction conditions were shown in Table 5 above to detect whether the cutting was correct. The PCR amplification results of normal uncut Neuro-2a cells and Neuro-2a positive cells with GDF11 gene knocked out were shown in Figure 4. As can be seen from Figure 4, compared with the length of the normal uncut PCR product, the length of the PCR product was significantly shortened after the GDF11 gene was knocked out, which proved that GDF11 in Neuro-2a cells was knocked out at the genomic level.

(2) Some monoclonal puromycin-resistant Neuro-2a positive cells were collected, and immunofluorescence staining was performed on normal uncut Neuro-2a cells and Neuro-2a positive cells with GDF11 gene knockout, respectively. The results are shown in FIG5 . As can be seen from FIG5 , Neuro-2a positive cells with GDF11 gene knockout did not express GDF11, which proved that GDF11 in Neuro-2a cells was knocked out at the protein level.

(3) Some monoclonal puromycin-resistant Neuro-2a positive cells were collected, and the normal uncut Neuro-2a cells and the GDF11 gene knockout Neuro-2a positive cells were stained with DAPI, and statistical analysis was performed. The results are shown in Figure 6. As can be seen from Figure 6, the nuclei of the GDF11 gene knockout Neuro-2a positive cells are enlarged relative to the nuclei of the normal uncut Neuro-2a cells. Cell aging generally results in enlarged nuclei, indicating that the GDF11 gene knockout Neuro-2a cells have aging characteristics.

(4) Collect some monoclonal puromycin-resistant Neuro-2a positive cells, and use Senescence β-Galactosidase Staining Kit (SA-β-Gal, Beyotime) to detect the activity of senescence-related β-galactosidase in normal uncut Neuro-2a cells and Neuro-2a positive cells with GDF11 gene knockout, respectively. The experimental steps are performed according to the instructions of the kit. Only when the cells are senescent, X-Gal as a substrate can be catalyzed by β-galactosidase to generate a blue product at pH 6.0; therefore, it is possible to judge whether the cells are senescent based on whether a blue product is generated, and then take photos with an Olympus BX53 microscope under the same parameters, and count the proportion of β-GAL positive cells; the results are shown in Figure 7. As can be seen from Figure 7, the number of Neuro-2a positive cells with GDF11 gene knockout increased significantly, indicating that knocking out GDF11 gene promotes cell senescence.

(5) The culture medium after sorting the puromycin-resistant Neuro-2a-positive cells was discarded, and the Neuro-2a-positive cells were fixed with a fixative (containing 2.5% glutaraldehyde and 4% paraformaldehyde (PFA)) for 10 min. The cells were gently scraped with a cell scraper and fixed in the same fixative at 4°C overnight. The normal uncut Neuro-2a cells and the Neuro-2a-positive cells with GDF11 knockout were prepared for transmission electron microscopy. The steps were as follows: a. Wash with 0.1 M PBS, 15 min/time, 3 times (centrifuge at 3000 rpm before each change of solution, and the following steps were the same); b. Fix with 1% natriuretic acid fixative for 1 h; c. ddH ₂ O washing, 15min/time, washing 3 times; d. 2% uranyl acetate fixation/staining for 30min; e. Gradient dehydration with 50%, 70%, 90%, and 100% ethanol in sequence, 15min/time, and then dehydration with 100% acetone twice, 20min/time; f. Pure acetone: embedding agent (volume ratio 3:1) infiltration at room temperature for 2h; g. Pure acetone: embedding agent (volume ratio 1:1) infiltration at room temperature for 2h; h. After embedding with pure embedding agent, place in an oven for polymerization: 37℃24h, 45℃24h, 60℃48h; i. Slice with an ultrathin microtome and stain with uranyl acetate-citrate, then observe and take pictures with a transmission electron microscope.

The transmission electron microscopy results are shown in Figure 8. As can be seen from Figure 8, compared with normal uncut Neuro-2a cells, the amount of lipofuscin in Neuro-2a positive cells with GDF11 gene knockout significantly increased, and the lipofuscin area significantly increased. Lipofuscin deposition is one of the manifestations of cell aging, indicating that GDF11 gene knockout promoted the aging of Neuro-2a cells.

The present invention provides a premature aging cell model established by targeted gene knockout, and its sgRNA, construction method and application. The dual sgRNA targeted knockout method is used to simultaneously generate two DNA double-strand break points in the second exon of the GDF11 gene and the intron sequence upstream thereof, and cuts the two sites on the second exon of the GDF11 gene, with high knockout efficiency. At the same time, the identification method is simple and the identification efficiency is high. Only PCR amplification and electrophoresis are required to determine whether the knockout is successful, which greatly reduces the identification cost after the knockout. A neuroblastoma cell line with GDF11 gene knockout, namely Neuro-2a cell line, is established, which provides an in vitro cell model for exploring the biological function of endogenous GDF11. Based on the experimental results, it is found for the first time that the Neuro-2a cell line with GDF11 gene knockout has aging characteristics, which provides a research tool for the research in the field of aging, promotes the research of anti-aging topics, and provides a theoretical research basis for delaying aging.

It should be noted that the embodiments of the present disclosure may be further described in the following manner:

A dual sgRNA for targeted knockout of the GDF11 gene, comprising GDF11-sgRNA1 and GDF11-sgRNA2; the targeting sequence of the GDF11-sgRNA1 is: GCCGAAGGTACACCCACAGT, (SEQ ID NO: 1) and the targeting sequence of the GDF11-sgRNA2 is: GAACCGGGTAAGGTAGCTTG. (SEQ ID NO: 2)

Optionally, the nucleotide sequence of the GDF11-sgRNA1 includes: GDF11-sgRNA1 Oligo1: 5'-CACCGGCCGAAGGTACACCCACAGT-3', (SEQ ID NO: 3) GDF11-sgRNA1 Oligo2: 5'-AAACACTGTGGGTGTACCTTCGGCC-3'; (SEQ ID NO: 4)

The nucleotide sequence of the GDF11-sgRNA2 includes: GDF11-sgRNA2 Oligo1: 5'-CACCGGAACCGGGTAAGGTAGCTTG-3', (SEQ ID NO: 5) GDF11-sgRNA2 Oligo2: 5'-AAACCAAGCTACCTTACCCGGTTCC-3'. (SEQ ID NO: 6)

A DNA molecule used to encode the double sgRNA for targeted knockout of the GDF11 gene.

A method for constructing a Neuro-2a cell line with a GDF11 gene knocked out comprises knocking out the GDF11 gene of the Neuro-2a cell line using a CRISPR/Cas9 system, wherein the double sgRNA used in the CRISPR/Cas9 system is a double sgRNA that targets the knockout of the GDF11 gene.

Optionally, the method for constructing Neuro-2a cell line with targeted knockout of GDF11 gene comprises:

Step 1, designing the targeting sequence of the dual sgRNA according to the GDF11 gene, and obtaining the nucleotide sequences of the dual sgRNA respectively;

Step 2, connect the nucleotide sequences of the dual sgRNAs to the pX459 vector respectively to obtain the expression vector of the dual sgRNAs;

Step 3, culturing the Neuro-2a cell line, and co-transfecting the expression vectors of the dual sgRNAs into the Neuro-2a cell line;

Step 4, screening, enriching, sorting and culturing to obtain puromycin-resistant Neuro-2a positive cells;

Step 5, extracting the genome of the puromycin-resistant Neuro-2a positive cell line and performing PCR amplification;

Step 6: Sequence the PCR amplification product and compare the sequencing result with the normal uncut reference sequence SEQ ID NO: 9 to determine whether it is correctly cut.

Optionally, the pX459 vector is used in knocking out the GDF11 gene of the Neuro-2a cell line using the CRISPR/Cas9 system.

Optionally, the dual sgRNAs are ligated to the vector pX459 by:

The sequences SEQ ID NO: 3 and SEQ ID NO: 4 were connected to the first pX459 vector after BbsI digestion to construct the expression vector pX459-U6-1-sgRNA-CMV-Cas9-T2A-PuroR; the sequences SEQ ID NO: 5 and SEQ ID NO: 6 were connected to the second pX459 vector after BbsI digestion to construct the expression vector pX459-U6-2-sgRNA-CMV-Cas9-T2A-PuroR.

A Neuro-2a cell line with GDF11 gene knocked out is obtained according to a method for constructing a Neuro-2a cell line with GDF11 gene knocked out.

A primer pair for identifying the mutation type of Neuro-2a cell line with GDF11 gene knocked out, the primer pair comprising: primers having nucleotide sequences as shown in SEQ ID NO: 7 and SEQ ID NO: 8.

Application of Neuro-2a cell line with GDF11 gene knockout in preparing a model for studying the function of the GDF11 gene.

Application of Neuro-2a cell line with GDF11 gene knockout in the preparation of disease models of cell senescence.

Application of Neuro-2a cell line with GDF11 gene knockout in preparing disease models of neuronal cell aging.

Application of Neuro-2a cell line with GDF11 gene knockout in screening drugs for treating or preventing cellular senescence.

Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present disclosure (including the claims) is limited to these examples. Based on the concept of the present disclosure, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the embodiments of the present disclosure as described above, which are not provided in detail for the sake of simplicity.

The embodiments of the present disclosure are intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the embodiments of the present disclosure should be included in the scope of protection of the present disclosure.

SEQUENCE LISTING

<110> Zhejiang University

<120> A premature aging cell model established by targeted gene knockout and its sgRNA, construction method and application

<130> FI210554

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<170> PatentIn version 3.5

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gttcaccaag gtactgaagg cccaactgtg ggtgtacctt cggcctgtgc cccgcccagc 720

cacagtctac ctgcagatct tacgactgaa acccctaact ggggaaggga ccgcaggggg 780

agggggtgga ggccggcgtc acatccgtat ccgttcacta aagattgagc tacactcacg 840

ttccggccac tggcagagca tcgacttcaa gcaagtgcta cacagctggt ttcgccagcc 900

acagagcaac tggggaatcg agatcaacgc ctttgatccc agcggcacag acctggctgt 960

cacctccctg gggccaggag ctgaggggct ggtgagcagg gggcctgaag tggtggtgga 1020

ggatatgtat aacctgtccc caagagatgg gagttagaaa aagtagatca ggaatgtggt 1080

ggttggggcc aggaactaat ttcagaggag gtggggtagc aactatcact tttcagggat 1140

ttaagccaga tgacagctga aaactaaaac tggatttggg gtgaaggtat cagttagtgg 1200

gagatccgtg ggaacacaaa gctgaccctt gggcgtggtg ttatctaatc ctcgcagtag 1260

ggaggccaga caggagggcc aggagctcta gaccgttacc tttgctacat agcaaatctg 1320

aggtcaacct gagcgacatg agagcccatc tcaaaacaaa acagaacctg gcccttgaca 1380

aggcgctggg ccaagaaccc gatgaggtca cattgccaga aaaggaagaa ttagggaaag 1440

atcctccacc accacttcag 1460

Claims (4)

1. A use of a Neuro-2a cell line with a GDF11 gene knocked out in preparing a disease model of neuronal cell aging, characterized in that the GDF11 gene of the Neuro-2a cell line is knocked out using a CRISPR/Cas9 system, wherein the CRISPR/Cas9 system uses a double sgRNA that targets the knockout of the GDF11 gene; wherein the double sgRNA includes GDF11-sgRNA1 and GDF11-sgRNA2; The targeting sequence of the GDF11-sgRNA1 is: GCCGAAGGTACACCCACAGT, The targeting sequence of the GDF11-sgRNA2 is: GAACCGGGTAAGGTAGCTTG. 2. The use according to claim 1, characterized in that the nucleotide sequence of the GDF11-sgRNA1 is: GDF11-sgRNA1 Oligo1: 5'—CACCGGCCGAAGGTACACCCACAGT—3', GDF11-sgRNA1 Oligo2: 5'—AAACACTGTGGGTGTACCTTCGGCC—3'; The nucleotide sequence of the GDF11-sgRNA2 is: GDF11-sgRNA2 Oligo1: 5'—CACCGGAACCGGGTAAGGTAGCTTG—3', GDF11-sgRNA2 Oligo2: 5′—AAACCAAGCTACCTTACCCGGTTCC—3′. 3. Use of a Neuro-2a cell line with a GDF11 gene knocked out in screening drugs for treating or preventing neuronal cell aging, characterized in that the GDF11 gene of the Neuro-2a cell line is knocked out using a CRISPR/Cas9 system, wherein the CRISPR/Cas9 system uses a dual sgRNA that targets the knockout of the GDF11 gene; wherein the dual sgRNA comprises GDF11-sgRNA1 and GDF11-sgRNA2; The targeting sequence of the GDF11-sgRNA1 is: GCCGAAGGTACACCCACAGT, The targeting sequence of the GDF11-sgRNA2 is: GAACCGGGTAAGGTAGCTTG. 4. The use according to claim 3, characterized in that the nucleotide sequence of the GDF11-sgRNA1 is: GDF11-sgRNA1 Oligo1: 5'—CACCGGCCGAAGGTACACCCACAGT—3', GDF11-sgRNA1 Oligo2: 5'—AAACACTGTGGGTGTACCTTCGGCC—3'; The nucleotide sequence of the GDF11-sgRNA2 is: GDF11-sgRNA2 Oligo1: 5'—CACCGGAACCGGGTAAGGTAGCTTG—3', GDF11-sgRNA2 Oligo2: 5′—AAACCAAGCTACCTTACCCGGTTCC—3′.