CN108486108B - Cell strain for knocking out human HMGB1 gene and application thereof - Google Patents

Abstract

The invention discloses a cell line knocking out human HMGB1 gene and its application, belonging to the field of molecular biology. The present invention provides a gRNA sequence for targeted knockout of human HMGB1 gene, the nucleotide sequence of which is shown in SEQ ID NO: 1. Using CRISPR-Cas9 technology to knock out the HMGB1 gene on the human Huh7 cell genome for the first time, the expression of HMGB1 protein was completely lost, and the HMGB1 knockout Huh7 cell line was obtained. It is an ideal HMGB1 knockout cell model; it is easy to operate, has a short cycle, low cost, and the modified cell line is stable. It can be used to study the role of HMGB1 protein in autophagy, apoptosis, cell inflammation, virus infection, etc. specific mechanism of action. The cell line obtained by the invention can significantly enhance the infection of the Japanese encephalitis virus strain.

Description

A cell line knocking out human HMGB1 gene and its application

technical field

The invention belongs to the field of molecular biology, and relates to a cell line knocking out human HMGB1 gene and its application.

Background technique

CRISPR (Clustered regularly interspaced short palindromic repeats) is a unique immune system evolved by bacteria in order to remove the foreign invasion genes of viruses, the CRISPR system. Using this system, bacteria can cut viral genes from their own chromosomes. In recent years, scientists have analyzed the key protein (Cas9) of this system from bacteria, and mastered the operation technology of this key protein. The complex with this key protein as the core can, under the guidance of a piece of RNA, search for the target DNA sequence, and then edit the DNA to disturb the gene or insert the desired sequence. CRISPR methods are rapidly surpassing zinc-finger nucleases and other editing tools, capable of rapidly trimming, cutting, replacing or adding biological DNA sequences with simple and low cost.

High-mobility group proteins (HMG proteins), widely present in eukaryotic cells, are named for their high mobility in polypropylene gel electrophoresis. HMG proteins are the drivers of gene regulation in eukaryotic cells, and are the most abundant group of chromatin proteins after histones in eukaryotic cells. They play an important role in the structure and function of chromatin and the regulation of gene expression. effect. The HMG protein family can be divided into three subfamilies: HMGA, HMGB and HMGN. High mobility group box 1 (HMGB1) belongs to the HMGB protein subfamily and is a DNA-binding protein that exists in the nucleus and cytoplasm. In the nucleus, HMGB1 is involved in various functions such as DNA replication, repair, recombination, transcription, and maintenance of genome stability. Extracellular HMGB1 plays an important role in inflammation, immunity, cell growth, proliferation, and death. In addition to its intranuclear and extracellular functions, HMGB1 in the cytoplasm can also bind to various proteins, participate in autophagy, cancer progression, and possibly non-traditional secretory pathways. When infected by external bacteria (endotoxins or intrinsic pro-inflammatory factors, etc.), macrophages and monocytes can actively release HMGB1; while necrotic or damaged cells passively release HMGB1 due to cell lysis.

SUMMARY OF THE INVENTION

In order to overcome the shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide a gRNA sequence for targeted knockout of the human HMGB1 gene.

Another object of the present invention is to provide a CRISPR/Cas9 lentiviral system for targeted knockout of human HMGB1 gene.

Another object of the present invention is to provide the application of the above-mentioned CRISPR/Cas9 lentivirus system for targeting the knockout of the human HMGB1 gene.

Another object of the present invention is to provide a cell line knocking out the human HMGB1 gene.

Another object of the present invention is to provide the application of the above-mentioned human HMGB1 gene knockout cell line.

The object of the present invention is achieved through the following technical solutions:

The present invention provides a gRNA sequence for targeting the knockout of human HMGB1 gene, the nucleotide sequence of which is: 5'-GAGTATCGCCCAAAAATCAA-3', SEQ ID NO: 1; it is located in the 322nd ~ 341 bits.

A CRISPR/Cas9 lentiviral system for targeting the knockout of the human HMGB1 gene, comprising the above-mentioned gRNA sequence for targeting the knockout of the human HMGB1 gene.

The construction method of the CRISPR/Cas9 lentivirus system for targeting and knocking out the human HMGB1 gene includes the following steps:

(1) Design gHMGB1-F and gHMGB1-R according to the above-mentioned gRNA sequence of targeted knockout of human HMGB1 gene, and anneal and phosphorylate gHMGB1-F and gHMGB1-R to form a fragment gHMGB1 containing sticky ends;

(2) Use BsmBI to digest the CRISPR/Cas9 lentiviral vector LentiCRISPRv2 to obtain the LentiCRISPRv2 vector after enzyme digestion;

(3) The gHMGB1 in step (1) is connected with the LentiCRISPRv2 vector after restriction enzyme digestion to obtain a CRISPR/Cas9 lentivirus system targeting the knockout of the human HMGB1 gene, which is named LentiCRISPRv2-HMGB1.

gHMGB1-F: 5′-caccgGAGTATCGCCCAAAAATCAA-3′,

gHMGB1-R: 5′-aaacTTGATTTTTGGGCGATACTCc-3′;

The application of the CRISPR/Cas9 lentivirus system targeting the knockout of the human HMGB1 gene in the preparation of a cell line for the knockout of the human HMGB1 gene. Preferably, the application is in the preparation of a human Huh7 cell line knocking out the human HMGB1 gene.

A cell line for knocking out the human HMGB1 gene is obtained by transfecting the above-mentioned CRISPR/Cas9 lentivirus system targeting the knocking out of the human HMGB1 gene into a target cell line. The cell line is a HMGB1 gene-silenced cell line, that is, the cell line does not express the HMGB1 protein at all.

A cell line knocking out the human HMGB1 gene is constructed through the following steps:

(1) packaging the above-mentioned CRISPR/Cas9 lentiviral system targeting the knockout of the human HMGB1 gene through packaging cells to obtain lentiviral particles;

(2) Infecting the target cell line with lentiviral particles to obtain a cell line knocking out the human HMGB1 gene.

Preferably, the target cell line is human Huh7 cell line.

Specifically include the following steps:

1) Packaging lentiviral particles on 293T cells

293T cells were transfected with the lentivirus packaging plasmid LentiCRISPRv2-HMGB1:psPAX2:pMD2.G=2:2:1, and the 293T packaging lentivirus supernatant was collected and centrifuged to obtain a suspension containing lentiviral particles;

2) Huh7 cells were infected with lentivirus

Infect Huh7 cells, the volume of DMEM complete medium: the volume ratio of the suspension containing lentiviral particles=1～3:1; preferably 2:1;

3) Puromycin screening knockout cell line

After the collected lentivirus was inoculated into Huh7 cells and cultured for 24 hours, an effective concentration of puromycin was added, and the culture was continued until the cells became full and then passaged; the passaged cells were continued to be screened with puromycin, and the culture medium was replaced every 2 days, and the cells were screened for 3 to 5 generations. ; After identification, a human Huh7 cell line with knockout of the human HMGB1 gene was obtained.

In step 3), the concentration of puromycin added according to the sensitivity of Huh7 cells is 1-2 μg/mL; preferably 1.5 μg/mL.

The application of the cell line knocking out the human HMGB1 gene in virus propagation.

The virus is Japanese encephalitis virus.

The application of the cell line knocking out the human HMGB1 gene in the study of JE.

JE virus SA14 strain can proliferate on this cell line, and the TCID ₅₀ of JE B virus SA14 strain is significantly higher than that of normal cell line after this cell line is infected with JE B virus SA14 strain.

The JE is Japanese encephalitis (JE for short).

The mechanism of the present invention is:

Huh7 is a passaged cell line of human liver cancer. Using the CRISPR/Cas9 system, the HMGB1 gene was knocked out in Huh7 cells to lose the expression of this protein. specific mechanism of action. The Huh7 cell line with HMGB1 knockout has not been reported before, and the knockout cell line screened by the present invention can be stably passaged, and the knockout cell line has no obvious difference from the control cells in terms of cell shape, growth rate, etc. , can be used as an ideal cell model. The HMGB1 gene knockout human Huh7 cell line disclosed in the present invention has been proved to be able to significantly enhance the infection of the Japanese encephalitis virus SA14 strain.

Compared with the prior art, the present invention has the following advantages and effects:

1) Using CRISPR-Cas9 technology to knock out the HMGB1 gene on the genome of human Huh7 cells for the first time, so that the expression of the HMGB1 gene is completely lost, and the Huh7 cell line with HMGB1 knockout is obtained. The operation is simple, the cycle is short, the cost is low, and the transformed cells The strain is stable and can be used to study the specific mechanism of HMGB1 protein in autophagy, apoptosis, cell inflammation, virus infection, etc.;

2) The HMGB1 protein was found to have been knocked out from both the gene and protein levels, indicating that the HMGB1 protein sequence had been completely changed, resulting in a complete loss of HMGB1 function, and the shape and growth rate of the knockout cell line were comparable to those of the control. There is no significant difference in cells, which is an ideal cell model for HMGB1 knockout;

3) Japanese encephalitis virus can proliferate in large quantities on this cell strain, after this cell strain is infected with Japanese encephalitis virus strain, compared with normal cell strains, its TCID ₅₀ is significantly elevated; can significantly enhance Japanese encephalitis virus strain infection.

Description of drawings

Figure 1 is a map of the target fragment gHMGB1 inserted into the LentiCRISPRv2 vector.

Figure 2 is a diagram of the sequencing results of the constructed LentiCRISPRv2-HMGB1 plasmid.

Figure 3 is a picture of frameshift mutations caused by insertion and deletion in knockout cell line sequencing.

Figure 4 is a WB detection chart of the expression of HMGB1 protein in knockout cell lines and control cell lines.

Figure 5 is a graph showing no significant difference in morphology between the knockout cell line and the control cell line.

Figure 6 shows that the TCID ₅₀ of the knockout cell line infected with the Japanese encephalitis SA14 strain was significantly higher than that of the control cell line.

Detailed ways

The present invention will be described in further detail below with reference to the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

The experimental method of unreceipted specific conditions in the embodiment, usually according to conventional conditions, such as molecular cloning experimental manuals such as Sambrook (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or according to the conditions suggested by the manufacturer's instructions.

Example 1 Construction of HMGB1 gene knockout lentiviral particles

1) Construction of LentiCRISPRv2-HMGB1 plasmid

The gRNA sequence of the target HMGB1 gene was selected according to The genome-scale CRISPR knock-out (GeCKO) libraries: 5′-GAGTATCGCCCAAAAATCAA-3′. According to LentiViral CRISPR Toolbox, the gRNA of HMGB1 gene was cloned into LentiCRISPRv2 (purchased from addgene, catalog number #98290) (Figure 1), and the constructed plasmid was named LentiCRISPRv2-HMGB1. The coding sequence of HMGB1 gene is shown in SEQ ID NO: 4, wherein, HMGB1CDS:CCDS9335.1.

Design and synthesize upstream and downstream primer sequences according to the sequence characteristics of the ligated ends of the LentiCRISPRv2 vector:

gHMGB1-F: 5′-caccgGAGTATCGCCCAAAAATCAA-3′,

gHMGB1-R: 5′-aaacTTGATTTTTGGGCGATACTCc-3′;

It was annealed and phosphorylated according to a fixed program to form a fragment gHMGB1 (25bp) containing sticky ends.

Annealing system (50 μL):

component Dosage H₂O 30μL Annealing Buffer for DNA oligos (5×) 10μL gHMGB1-F 5μL gHMGB1-R 5μL

Annealing program: 95°C, 5min, drop once every minute to 25°C.

The fragment gHMGB1 containing sticky ends was connected to the LentiCRISPRv2 vector after BsmBI digestion, and the ligation product was transformed into Stbl3 competent cells (the use of Stbl3 competent cells was carried out according to the commercial instructions, and Stbl3 was purchased from Beijing Quanshijin Biotechnology Co., Ltd., Item number CD512-01) was cultured at 37°C overnight, and multiple positive clones were obtained by PCR electrophoresis of bacterial liquid. The sequencing results showed that the target sequence was successfully connected to the vector (Figure 2).

The correct positive clones were inoculated into the LB liquid medium with Amp ⁺ resistance at a ratio of 1:500, placed in a shaker at 37 °C, and incubated at 180 r/min for 14-16 h. Follow the instructions of EZNA Endo-free Plasmid Midi Kit II. The LentiCRISPRv2-HMGB1 plasmid was obtained by extracting the endotoxin-depleted plasmid.

Enzyme digestion system (50μL):

component Dosage LentiCRISPRv2 2μg BsmBI 2μL Buffer(10×) 5μL H₂O to 50μL

Reaction conditions for digestion: 55°C for 2h.

Ligation system (20 μL):

component Dosage The digested vector 2μL T4 ligase 2μL T4 Buffer 2μL gHMGB1 14μL

Ligation reaction conditions: ligation overnight at 16°C.

2) Lentiviral particle packaging

293T cells (purchased from the Shanghai Chinese Academy of Sciences Cell Bank) were plated into 60 mm cell culture dishes, and the cells were grown to 70-80% for transfection. Cells were exchanged into 1 mL of pre-warmed serum-free medium (DMEM) prior to transfection. First, add 6μg LentiCRISPRv2-HMGB1 expression vector plasmid, 6μg psPAX2 (purchased from addgene, catalog #12260) and 3 μg pMD2.G (purchased from addgene, catalog #12259) helper plasmids into 1 mL of DMEM, and mix well. Then, the 100 μM PEI (polyethyleneimine) stock solution was diluted to 10 μM with HBS (Balanced Salt Solution of HBS) and mixed well. Then, 100 μL of the diluted 10 μM PEI solution was added to the above-mentioned DMEM containing the plasmid, mixed well, and then allowed to stand at room temperature for 5-10 min. Finally, this mixture was added dropwise to the 293T cells to be transfected, the dish was shaken gently to mix well, and then incubated in a 37°C incubator with 5% CO ₂ . After 3 h, it was replaced with 4 mL of DMEM pre-warmed at 37°C. After culturing for 48 hours, the cell culture medium containing lentivirus was collected, centrifuged at 7000 r/min at 4°C for 15 minutes, and the obtained supernatant was the suspension containing lentivirus particles.

Example 2 Huh7 infected lentiviral particles and screened to obtain cell lines

1) Determination of the optimal effective concentration of Puromycin

Huh7 cells (purchased from the Shanghai Chinese Academy of Sciences Cell Bank) were seeded in 12-well plates at a concentration of 2×10 ⁵ cells/mL. After culturing for 24 hours, puromycin was treated at concentrations of 0, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8, 2.1 μg/mL fresh anti-serum-free medium solution replaces the old medium in each well. The cell viability was observed daily, and the fresh antiserum-free medium solution containing puromycin was replaced every two days according to the cell viability. The optimal effective concentration is the concentration that can kill all cells within 3 to 5 days. The optimal effective concentration measured on Huh7 cells in this experiment was 1.5 μg/mL.

2) Lentiviral particle infection of Huh7 screening cell line

The lentiviral particles collected in Example 1 were inoculated into Huh7 cells, and after culturing for 24 hours, the optimal effective concentration of puromycin 1.5 μg/mL was added, and the cells were further cultured until the cells became confluent and then passaged. Use puromycin to continue to screen the passaged cells, change the culture medium every 2 days, and screen for 3 to 5 generations.

Example 3 Identification of HMGB1 gene knockout cell lines

1) Identification of genomic DNA sequencing:

The genomic DNA of the cell line was extracted according to the instructions of the EZNA Tissue DNA Kit. Primers were designed at 200-300 bp upstream and downstream of the gene sequence region targeted by the gRNA, the fragment was amplified by PCR, cloned into pMD ^TM 19-TVector (Takara), and the positive plasmid was selected and sent to Yingweijieji (Shanghai) Trading Co., Ltd. The company conducts sequencing.

The sequence alignment results showed that the target site had frameshift mutations due to insertion or deletion of bases (Figure 3). Denoted as cell line 1, cell line 2, and cell line 3; among them, cell line 1 has a 5-base deletion in the targeted knockout site of the wild-type HMGB1 gene; cell line 2 is in the wild-type HMGB1 gene. One base was inserted into the targeted knockout site; cell line 3 had a six-base deletion in the targeted knockout site of the wild-type HMGB1 gene.

The specific knockout sites of the HMGB1 gene are as follows, and the following sequences correspond in sequence: wild type, cell line 1, cell line 2, and cell line 3:

TGCTCT GAGTATCGCCCAAAAATCAAAGGAGA ;

TGCTCT GAGTATCGCCCAAAAA----- GGAGA;

TGCTCT GAGTATCGCCCAAAAATTCAA AGGAGA;

TGCTCT GAGTATCGCCCAAAAA------- GAGA.

2) Western Blot (WB) to verify gene protein knockout effect:

①Protein glue preparation: After washing and drying the glass plate, put it in the clip and clamp it, and then clamp it vertically on the shelf to prepare for glue filling. Prepare 10% or 15% separating gel according to the ratio in Table 1, add TEMED (tetramethylethylenediamine), shake well and pour the gel immediately, until the surface of the glue reaches the height of the middle line of the green belt; add isopropyl alcohol to the glue Liquid seal. After the separating gel is fully solidified, the isopropanol layer on the gel is poured off and blotted dry with filter paper. Prepare 5% stacking gel according to Table 2, add TEMED, shake well and pour the gel immediately; after the remaining space is filled with stacking gel, insert the comb into the stacking gel, and when the stacking gel solidifies, gently pull out the comb. Store in an airtight refrigerator at 4°C.

Table 1 formula of separating gel

component 10% (5mL) 15% (5mL) H₂O 1.9mL 1.1mL 30% Acrylamide 1.7mL 2.5mL 1.5M Tris-HCl (pH8.8) 1.3mL 1.3mL 10% SDS 50μL 50μL 10% Ammonium Persulfate 50μL 50μL TMEMD 2μL 2μL

Table 2 Stacking Gum Formulation

component 5% (3mL) H₂O 2.1mL 30% Acrylamide 0.5mL 1.5M Tris-HCl (PH6.8) 380μL 10% SDS 30μL 10% Ammonium Persulfate 30μL TMEMD 3μL

②Sampling and electrophoresis: After the protein gel is clamped into the electrophoresis tank, add 1×SDS-PAGE electrophoresis buffer to the electrophoresis tank, and add 5μL Protein Ladder and 20μL protein sample in sequence to the sample hole of the protein gel. Connect the device correctly, electrophoresis at 80V for 30min, and electrophoresis at 120V for 60min. When the bromophenol blue just ran out of the protein gel, the electrophoresis was stopped and the membrane was transferred.

③ Transfer membrane: Cut the NC membrane and filter paper according to the size of the protein glue, put them in the membrane transfer solution and soak for 10 minutes. Place the three layers of filter paper, protein glue, NC membrane, and three layers of filter paper on the inner side of the transfer clip in the order. Fasten the transfer clip and put it into the film transfer machine in the correct direction, that is, the protein glue is located at the negative electrode, and the NC membrane is located at the positive electrode. The membrane transfer instrument was placed on ice, and the membrane was transferred under a constant current of 200 mA for 55 min.

800CWGOAT Anti-RABBIT IgG(H+L)购自LICOR化学免疫试剂公司)，室温孵育1h。回收二抗，TBST缓冲液洗涤NC膜三遍，每遍10min。使用Odyssey双色红外激光成像系统扫描NC膜成像。④Immunoblotting: After transfer, remove the NC membrane, wash the NC membrane with TBST buffer, and add blocking solution containing 5% nonfat milk powder to block for 2 hours at room temperature or overnight at 4°C. The blocking solution was discarded, and the NC membrane was washed three times with TBST buffer for 10 min each time, and the diluted primary antibody (HMGB1 rabbit polyclonal antibody was purchased from Abcam, product number ab18256; β-Actin rabbit polyclonal antibody was purchased from Quanzhou Gold Co., Ltd. , Cat. No. HC201-01), incubated for 2h at room temperature or overnight at 4°C. The primary antibody was recovered, the NC membrane was washed three times with TBST buffer for 10 min each time, and a 1:10000 diluted goat anti-mouse fluorescent secondary antibody (

800CWGOAT Anti-RABBIT IgG (H+L) was purchased from LICOR Chemical Immunoreagent Company) and incubated at room temperature for 1 h. The secondary antibody was recovered, and the NC membrane was washed three times with TBST buffer for 10 min each time. The NC films were scanned using the Odyssey two-color infrared laser imaging system.

The cells were lysed with Biyuntian's lysate, and the cell lysate of the cell line was collected. The detection results showed that the expression of HMGB1 protein had been completely knocked out (Fig. 4). Among them, KO-HMGB1 in Fig. 4 represents a HMGB1 knockout cell line.

Example 4 Observation of cell morphology of HMGB1 gene knockout cell line

During the continuous passage of the HMGB1 gene knockout cell line (KO-HMGB1) and the Huh7 normal cell line at the same time, there was no significant difference in morphology between the two (Figure 5). After serial passage, the expression of HMGB1 protein in the cells was detected and the genome sequenced, and it was found that the constructed cell line was very stable. It is not difficult to imagine that the knockout cell line has great application prospects.

Example 5 Functional verification of HMGB1 gene knockout cell line on JE

HMGB1 knockout cell line (KO-HMGB1) and Huh7 normal cell line were inoculated in 12-well plates, respectively. Document "Biology and molecular characteristics of a strong neurovirulent Japanese encephalitis virus strain. Acta Virology. 2010, 4(26), 265-270" published in inoculation). 48h after virus inoculation, the cell supernatant was collected and stored in a -80°C refrigerator.

The virus supernatant was inoculated into a 96-well plate of 70-80% BHK-21 (purchased from the cell bank of Shanghai Chinese Academy of Sciences) monolayer cells. After 48 hours, DMEM was discarded, washed twice with PBS buffer, and 50 μL of pre-cooling was added to each well. Methanol, placed at -20 °C for 30 min; discarded methanol, washed three times with PBS buffer, added 50 μL of primary antibody (JEV NS1 protein monoclonal antibody, custom-made by Abimat Biopharmaceutical Co., Ltd.) to each well, and incubated at 37 °C for 1 h ; Discard the primary antibody, wash three times with PBS buffer, add 50 μL of HRP-coupled secondary antibody (goat anti-mouse horseradish peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) to each well, cat. No. 115-035 -003), incubate at 37 °C for 30 min; discard the secondary antibody, wash with PBS buffer three times, add 50 μL of AEC color development working solution, and incubate at 37 °C for 30 min in the dark; after the color development is completed, discard the display solution and wash with PBS buffer for two times all over. Cell staining was observed under a microscope (Japanese encephalitis virus did not enter the nucleus), and TCID ₅₀ was counted and calculated.

The results showed that the virus titer in the knockout cell line was significantly increased (Fig. 6), indicating that Japanese encephalitis virus can proliferate in this cell line.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

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Claims (4)

1. The application of a cell line knocking out the human HMGB1 gene in the propagation of Japanese encephalitis virus, characterized in that: the cell line is a human Huh7 cell line. 2. application according to claim 1, is characterized in that: the cell line of described knockout of human HMGB1 gene is obtained by the CRISPR/Cas9 lentivirus system of targeted knockout of human HMGB1 gene transfecting target cell line; The CRISPR/Cas9 lentivirus system for targeting and knocking out the human HMGB1 gene contains a gRNA sequence for targeting and knocking out the human HMGB1 gene; The gRNA sequence for the targeted knockout of the human HMGB1 gene is: 5'-GAGTATCGCCCAAAAATCAA-3'. 3. application according to claim 2, is characterized in that: The method for constructing a cell line knocking out the human HMGB1 gene comprises the following steps: (1) Packaging the CRISPR/Cas9 lentivirus system targeting the knockout of the human HMGB1 gene through packaging cells to obtain lentiviral particles; (2) Infect the target cell line with lentiviral particles to obtain a cell line knocking out the human HMGB1 gene. 4. application according to claim 2, is characterized in that: The construction method of the CRISPR/Cas9 lentivirus system for targeting and knocking out the human HMGB1 gene includes the following steps: (1) Design gHMGB1-F and gHMGB1-R according to the above-mentioned gRNA sequences targeting knockout of human HMGB1 gene, and anneal and phosphorylate gHMGB1-F and gHMGB1-R to form a fragment gHMGB1 with sticky ends; gHMGB1-F: 5′-caccgGAGTATCGCCCAAAAATCAA-3′, gHMGB1-R: 5′-aaacTTGATTTTTGGGCGATACTCc-3′; (2) Use BsmBI to digest the CRISPR/Cas9 lentiviral vector LentiCRISPRv2 to obtain the digested LentiCRISPRv2 vector; (3) The gHMGB1 in step (1) was connected with the LentiCRISPRv2 vector after restriction enzyme digestion to obtain a CRISPR/Cas9 lentivirus system targeting the knockout of the human HMGB1 gene, which was named LentiCRISPRv2-HMGB1.

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