WO2019223039A1 - Sgrna screening for dj-1 gene editing, vector thereof and use thereof - Google Patents

Abstract

Provided is an sgRNA for DJ-1 gene, the sequence thereof being as shown in SEQ ID NO: 1. Further provided are a drug and a plasmid for DJ-1 gene, the drug and the plasmid comprising the sgRNA.

Description

Screening of sgRNA for DJ-1 gene editing and its vector and application Technical field

The invention belongs to gene technology, and particularly relates to sgRNAs targeted to the DJ-1 gene, has high editing efficiency, and includes sgRNAs and vectors and applications thereof.

Background technique

DJ-1 (PARK7) is a more common autosomal recessive Parkinson's disease-causing gene. Its pathogenic mechanism to Parkinson's disease is unclear and needs to be studied. CRISPR / Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats / Cas9) The gene editing system is a third-generation gene editing system developed from ZFNs and TALENs. It is discovered from the adaptive immune defense system of bacteria and used to combat foreign DNA and invasion. Viruses have been widely used in the field of biomedicine.

technical problem

Before the emergence of CRISPR / Cas9 technology, studying gene functions at the cellular level, the widespread use of RNA interference (RNAi) technology, can only reduce the expression of genes, but cannot completely knock them out at the DNA level. This is the biggest advantage of CRISPR / Cas9. During the application of CRISPR / Cas9, the level of sgRNA specificity largely determines the efficiency of CRISPR / Cas9 editing.

Technical solutions

The invention discloses an optimal sgRNA of a DJ-1 gene, which provides a reference for future gene therapy.

The present invention adopts the following technical solutions:

A sgRNA directed to the DJ-1 gene, the sequence of the sgRNA directed to the DJ-1 gene is SEQ ID NO.1.

A drug targeting the DJ-1 gene includes the sgRNA shown in SEQ ID NO.1.

In the above technical solution, the drug targeting the DJ-1 gene further includes a drug carrier, such as a conventional polymer carrier, a cell carrier, and the like.

A plasmid targeting the DJ-1 gene includes the sgRNA shown in SEQ ID NO.1.

Preferably, the plasmid targeting the DJ-1 gene is a pSpCas9 (BB) -2A-GFP plasmid loaded with the sgRNA shown in SEQ ID NO.1.

Application of the sgRNA shown in SEQ ID NO. 1 in the preparation of a drug directed against the DJ-1 gene.

Application of the sgRNA shown in SEQ ID NO. 1 in the preparation of a drug for treating or relieving Parkinson's disease.

In the present invention, the prokaryotic evaluation system such as a bluish blue clone formation experiment confirms that the editing efficiency of the above sequence reaches 75%.

First, the partial sequence encoding the β-gal region on the pMD-19T plasmid was replaced by a long sequence containing the target sequence, thereby forming a frameshift mutation. The replaced plasmid was called pMD-repeat plasmid, which was transformed into the plasmid containing X-gal and Blue colonies cannot be formed in the solid medium of IPTG; when co-transformed with the pCas9 plasmid loaded with the target sequence corresponding sgRNA, the target sequence in the pMD-repeat plasmid will be cleaved by Cas9 guided by the sgRNA, and the target sequence will have two repeating sequences Homologous recombination occurred, and the gene sequence encoding β-gal was restored from frameshifting to non-frameshifting state. Under the induction of X-gal and IPTG, blue colonies were formed.

The prokaryotic gene knockout pCas9 plasmid containing the sgRNA sequence and the pMD-repeat plasmid containing the corresponding target sequence were constructed. The two plasmids were co-transformed into DH5α competent cells in equal amounts. X-gal-TPTG-chloramphenicol-ampicillin Culture, observe the proportion of blue colonies in the total colonies. Construct a eukaryotic gene knockout pSpCas9 (BB) -2A-GFP plasmid containing sgRNA sequences, transfect HeLa cells, and perform gene editing. Then extract the cell genomes in the control and experimental groups, and design primers in the upstream and downstream regions of the sgRNA. Q5 high-fidelity enzyme PCR, product purification, T7E1 enzyme digestion, agarose gel electrophoresis, and observation of the band results after electrophoresis; design sequencing primers, sequence the purified product, and observe whether there are nesting peaks and nesting peaks near the Cas9 digestion site High and low. The data proves that the present invention discloses that the sgRNA directed to the DJ-1 gene has the best editing efficiency; after transfection into Hela cells, it will cause frame shifting of the gene reading frame, translation will be terminated early, and the DJ-1 gene will be knocked out.

Beneficial effect

After transfection of Hela cells, the DJ-1 gene was completely knocked out, which verified that the sgRNA of the present invention has excellent editing efficiency, and demonstrated the feasibility and effectiveness of constructing a monoclonal gene knockout cell line through cell proliferation experiments. After the DJ-1 gene knockout, the cell proliferation of the cell line is slowed down; it provides an effective cellular tool for the study of DJ-1 mechanism.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of the sgRNA clone of the pCas9 plasmid;

Figure 2 is a partial base sequence diagram of the lacZ gene of the pMD-repeat plasmid;

FIG. 3 is a sequence diagram of the target sequence corresponding to the sgRNA of the pMD-repeat plasmid;

Figure 4 is a schematic diagram of the experimental principle of the formation of albino blue clones;

FIG. 5 is a colony diagram of DJ-1-sgRNA dual-plasmid co-transformation colony formation experiment;

FIG. 6 is a graph showing the ratio of blue colonies to total colonies of the DJ-1-sgRNA dual plasmid co-transformation of the whitening blue clone formation experiment (n = 3, mean ± SD);

FIG. 7 is a sequence diagram of sgRNA4 sequence insertion of pSpCas9 (BB) -2A-GFP plasmid; FIG.

Figure 8 is a Western Blot diagram of a DJ-1 knockout monoclonal HeLa cell line;

FIG. 9 is an immunofluorescence image of a DJ-1 knockout monoclonal HeLa cell line; FIG.

Figure 10 is a graph showing the results of cell proliferation experiments.

Embodiments of the invention

The reagents are all commercially available products.

Examples

The sgRNA of the DJ-1 gene, the sequence of the sgRNA of the DJ-1 gene is SEQ ID NO. 1, specifically 5'- ACATCACGGCTACACTGTACTGG -3 ', referred to as sgRNA4.

Comparative example

Two other sgRNAs were selected for comparison, as follows:

SEQ ID NO.2: 5'- AGTACAGTGTAGCCGTGATGTGG -3 ', referred to as sgRNA1

SEQ ID NO. 3: 5'- CTGCACAGATGGCGGCTATCAGG -3 ', referred to as sgRNA2.

1. Construct a prokaryotic gene knockout CRISPR / Cas9 plasmid loaded with the three DJ-1 sgRNAs according to conventional methods, see Figure 1 for details; pCas9 plasmid digestion system (pCas9 plasmid 2 μg, BsaI enzyme (NEB) 2 μL, 100X BSA (NEB) 1 μL, 10X NEB Buffer 10 μL, ddH ₂ Oup to 100 μL), digested overnight at 37 ° C in a water bath. The digested product and undigested plasmid were identified by agarose gel electrophoresis at the same time. After the digestion, it was added to 1.2 % Agarose gel wells, run at 120V for about 30 minutes, and use 1kb DNA Marker as a reference. The gel is purified and recovered. The experimental steps for purification and recovery are as follows:

(1) Add 500µL of equilibration solution BL to the adsorption column, centrifuge at 12000rpm for 1min, discard the waste liquid, and reposition the adsorption column in the collection tube; (2) Cut the target band from the gel, and possibly remove excess clot Gel, cut the gel and weigh it; (3) Add an equal volume of PC to the gel (if the gel is 0.1g, the volume is considered as 100µL, and the added PC volume is 100µL), 56 ° C The water bath was kept for 10 minutes, and the mixture was continuously mixed upside down; (4) The dissolved liquid was cooled to room temperature, sucked into the adsorption column, centrifuged at 12000 rpm for 1 min, the waste liquid was discarded, and the adsorption column was placed in the collection tube again; (5) toward the adsorption column Add 600µL PW (anhydrous ethanol has been added to the rinsing solution PW), leave it to stand for 4min, and centrifuge at 12000rpm for 1min, discard the waste liquid, and place the adsorption column in the collection tube again. Repeat this operation (6). The collection tube was left at 12000 rpm for 2 minutes, and dried at room temperature for several minutes. (7) Take a new centrifuge tube, place the adsorption column, suck 30-50 µL ddH ₂ O into the adsorption column, and place it for 2 minutes and centrifuge at 12000 rpm for 2 minutes. The resulting solution is purified Cleavage product.

Phosphorylation of sgRNA sequences

Zh The Oligo synthesized by Jin Weizhi was diluted to 10 μM and phosphorylated. The Oligo sgRNA sequence is as follows:

AAACAGTACAGTGTAGCCGTGATG, AAAACATCACGGCTACACTGTACT; AAACCTGCACAGATGGCGGCTATCG, AAAACGATAGCCGCCATCTGTGCAG; AAACACATCACGGCTACACTGTACG, AAAACGTACAGTGTAGCCGTGATGT; the above pairs correspond to SEQ respectively ID NO.2, SEQ ID NO.3, SEQ ID NO.1; see Table 1 for phosphorylation system, 37 ° C, 30min.

Table 1 Phosphorylation system

Component volume Oligo F 10 μL Oligo R 10 μL T4 PNK enzyme 1 μL 10X T4 Ligase Buffer 5μL ddH ₂ O up to 50μL

Zh The sgRNA was annealed, and 2.5 μL of 1M sodium chloride was added to the phosphorylated product, followed by annealing with a PCR instrument for 2 h, and then slowly cooled from 95 ° C to room temperature, and the final product was diluted 10-fold.

3. Connection. The connection system is shown in Table 2. The system reacted at 16 ℃ overnight.

Table 2 Connection reaction system

ingredient volume Oligo products after annealing 2 μL PCas9 plasmid vector after digestion 1 μL T4 ligase (NEB) 1 μL 10X T4 Ligase Buffer (NEB) 2 μL ddH ₂ O up to 20μL

4.Conversion

(1) Take 50µL DH5α competent cells on ice, add the product at 20 ℃ after the above connection, mix gently and let stand for 30min; (2) heat-shock in a 42 ℃ water bath for 45sec, and quickly put on ice for 10min ; (3) 800 μL of antibiotic-free LB liquid culture medium, 37 ° C, 220 rpm, constant temperature shaking for 50 min; (4) in a clean bench, transfer the bacterial solution to the LB solid medium containing chloramphenicol with a pipette. , Allow to stand for 20min, and invert and incubate in a 37 ° C constant temperature incubator; 5) After 12 hours, pick 10 colonies to culture in LB liquid medium containing chloramphenicol, and extract the plasmid.

5.Extraction of plasmid DNA

Follow the instructions of Tiangen's small plasmid extraction kit (article number # DP103-03)

(1) Add 500 µL of equilibration solution BL to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and re-place the adsorption column in the collection tube; (2) 5 mL of bacterial solution cultured overnight, centrifuge at 12000 rpm for 15 min, and discard the supernatant (3 ) Add 250µL of solution P1 (included with the kit, RNaseA has been added) to a centrifuge tube containing bacterial cells, vortex to dissolve the precipitate; (4) add 250µL of solution P2 (included with the kit), and mix gently by turning it upside down. Evenly mix 8-10 times to lyse the bacteria; (5) immediately add 350µL solution P3 (supplied with the kit) and mix gently upside down for 10 times. At this time, white flocculent precipitates appear, and centrifuge at 12000rpm for 10min; (6) Transfer the supernatant from the centrifuge tube to the adsorption column with a pipette, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and place the adsorption column back into the collection tube; (7) Add 600 µL PW (rinsing solution PW) to the adsorption column. Anhydrous ethanol has been added), stand still for 2min, centrifuge at 12000rpm for 1min, discard the waste liquid, reposition the adsorption column in the collection tube, repeat this operation once; (8) empty the collection tube containing the adsorption column at 12000rpm for 2min, Dry at room temperature Min; (9) takes a new centrifuge tube, into the adsorption column, to absorb 100μL ddH ₂ O adsorption column, placed in 2min, centrifuged at 12000 rpm 2min, with a Nanodrop measured for concentration and purity of plasmid 2000.

The extracted plasmid was sent to Suzhou Jinweizhi Co. for sequencing to check whether the target fragment was inserted into the plasmid correctly.

6.Construction of recombinant pMD-repeat plasmid-target sequence

The pMD-repeat plasmid was modified from the pMD-19T plasmid. Using the HIV partial sequence as a reference, design a long sequence to replace the original sequence between the KpnI and HindIII digestion sites in the lacZ gene of pMD-19T plasmid. The long sequence contains two HIV repeats and one target sequence. The mutation of the original KpnI and HindIII restriction sites on the plasmid disappeared, and the KpnI and HindIII restriction sites between the two repeats and the target sequence can be used to insert the target sequence corresponding to the sgRNA. After the transformation, the reading frame of the plasmid lacZ gene is frame-shifted to generate a stop codon and cannot form α-complement. It is called pMD-repeat plasmid. The base sequence of the reading frame of the lacZ gene of pMD-repeat plasmid is modified as shown in Figure 2. Part of the base sequence of the lacZ gene of pMD-repeat plasmid, the red box represents the HIV repeat sequence; the black box represents the target sequence, and the KpnI and HindIII digestion sites are located between the red box and the black box.

7, pMD-repeat plasmid digestion, gel purification and recovery

The long sequence contained in the pMD-repeat plasmid contains KpnI and HindIII digestion sites. Between the repeat sequence and the target sequence, the target sequence can be inserted after digestion. The pMD-repeat plasmid was digested with KpnI and HindIII. The digestion system (PMD-repeat plasmid 1 μg, Hind III enzyme 0.5 μL, Kpn I enzyme 0.5 μL, 10X NEB Buffer 2.1 2 μL, ddH ₂ O to 20 μL), 37 ° C water bath reaction 2h; after digestion, the product was identified by agarose gel electrophoresis, and the gel was purified and recovered.

Oligonucleotide complementary to sgRNA target sequence

The target oligonucleotides corresponding to the three DJ-1 sgRNAs synthesized by Suzhou Jinweizhi Company were complementary. The reaction system was: 10 × Anneal Buffer 2 μL, Oligo F 1 μL (10 μM), Oligo R 1 μL (10 μM), ddH ₂ O16 μL was added, and the total volume was 20 μL. The reaction conditions are: 95 ° C, 2min; 1 ° C to 65 ° C every 30sec; 65 ° C, 5min; 1 ° C to 25 ° C every 1min; 25 ° C, 1min, and then cooled to 4 ° C, the target sequence oligonucleotide chain The sequence is as follows:

AGCTTCAGTACAGTGTAGCCGTGATGTGGGGTAC, CCCACATCACGGCTACACTGTACTGA; AGCTTCCTGCACAGATGGCGGCTATCAGGGGTAC, CCCTGATAGCCGCCATCTGTGCAGGA; AGCTTCACATCACGGCTACACTGTACTGGGGTAC, CCCAGTACAGTGTAGCCGTGATGTGASEQ, corresponding to the above ID NO.2, SEQ ID NO.3, SEQ ID NO.1.

The purified and recovered pMD-repeat plasmid was ligated with the annealed complementary oligonucleotide strand. 1 μL of the digested pMD-repeat plasmid, Oligo 7.5 μ after annealing, 0.5 μL of T4 ligase (NEB), 10X T4 Buffer The reaction was performed at 1 μL overnight at 16 ° C. The target sequence corresponding to the pMD-repeat plasmid sgRNA was inserted and sequenced.

The ligated product was transformed into DH5α competent cells, 800 μL of LB liquid medium was added, and cultured at 37 ° C with shaking for 40 minutes. The plates were cultured on a plate containing ampicillin resistance at 37 ° C. After 12 hours, 5 colonies were picked and the The ampicillin was cultured in LB liquid medium, and the plasmid was extracted. The extracted plasmid was sent to Suzhou Jinweizhi Company for sequencing, and the target sequence was correctly inserted into the plasmid, and stored for future use.

8.Construction of recombinant pSpCas9 (BB) -2A-GFP plasmid-sgRNA

Construction of three eukaryotic gene knockout CRISPR / Cas9 plasmids loaded with DJ-1 sgRNA. pSpCas9 (BB) -2A-GFP plasmid digestion, gel purification and recovery, pSpCas9 (BB) -2A-GFP plasmid digestion system (pSpCas9 (BB) -2A-GFP plasmid 2μg, BbsI enzyme (NEB) 2μL, 100X BSA ( NEB) 1 μL, 10X NEB Buffer 2.110 μL, ddH ₂ O up to 100 μL), and reacted at 37 ° C in a water bath for 4 hours. After digestion, the product was identified by agarose gel electrophoresis, and purified by gel digestion and recovery.

Dilute Oligo synthesized by Jinweizhi Company to 10 μM, phosphorylate, Oligo sgRNA sequence is CACCGAGTACAGTGTAGCCGTGATG, AAACCATCACGGCTACACTGTACTC; CACCGCTGCACAGATGGCGGCTATC, AAACGATAGCCGCCATCTGTGCAGC; CACCGCACATCACGGGGACACTGTGACGCGTACACGTAGAC, ACGCGTACACGTAGAC ID NO.3, SEQ ID NO.1; see Table 3 for phosphorylation system, 37 ° C, 30min. The phosphorylated product was annealed with a PCR machine for 2h, 95 ° C, 5min; 1 ° C to 25 ° C every 1min; 25 ° C, 1min, and then cooled to 4 ° C. Slowly cool to room temperature, dilute the final product 10 times; then connect, connect the system as shown in Table 4, react at 16 ° C overnight; transform the connected product into DH5α competent cells, add 800 μL LB liquid medium, 37 ° C, shake for 40min Cultivate and culture on a plate containing ampicillin resistance at 37 ° C. After 12 hours, pick 5 colonies and culture in LB liquid medium containing ampicillin, extract the plasmid, and send the extracted plasmid to Suzhou Jinweizhi Company for sequencing and detection of the target. The sequence was inserted into the plasmid correctly and saved for future use.

Table 3 Phosphorylation system

Component volume Oligo F 10 μL Oligo R 10 μL T4 PNK enzyme 1 μL 10X T4 Ligase Buffer 5μL ddH ₂ O up to 50μL

Table 4 Connection reaction system

ingredient volume Oligo products after annealing 2 μL PSpCas9 (BB) -2A-GFP plasmid vector after digestion 1 μL T4 ligase (NEB) 1 μL 10X T4 Ligase Buffer (NEB) 2 μL ddH ₂ O up to 20μL

9 、 Album blue clone formation experiment

When the pCas9 plasmid containing the sgRNA sequence and the pMD-repeat plasmid containing the target sequence corresponding to the sgRNA co-transform DH5α competent cells, the Cas9 enzyme will recognize and cut the target sequence under the sgRNA mediation, causing the DNA double strand to break. Homologous recombination will occur between repeated sequences. Only one repeat sequence will have the reading frame of the lacZ gene. It will change from a frameshift state to a non-frameshift state. Under the induction of X-gal and IPTG, blue colonies will form. Experiment The schematic diagram is shown in Figure 4.

Co-transformation experiment

When the pCas9 plasmid containing the sgRNA sequence and the pMD-repeat plasmid containing the target sequence corresponding to the sgRNA were transformed into 50 μL of DH5α competent cells in equal amounts, 800 μL of LB liquid medium was added, and cultured at 37 ° C with shaking for 40 min. Cultivate TPTG-chloramphenicol-ampicillin on a plate at 37 ° C and observe the ratio of blue colonies to the total colonies. Pick blue colonies and cultivate for 12 h in LB liquid medium containing chloramphenicol-ampicillin resistance. Sequencing with the universal primers of pMD-19T to observe whether the target sequence was digested by enzymes and homologous recombination between repeated sequences occurred.

10. Results of whitening blue clone formation experiment

The pCas9 plasmid loaded with DJ-1 sgRNA and the corresponding pMD-repeat plasmid loaded with the target sequence were respectively co-transformed into DH5α competent cells in equal amounts, cultured on X-gal-IPTG-Cl-Amp plates, and repeated three times, representatively The colony growth chart is shown in Figure 5. According to the colony map of the three sgRNAs of DJ-1, it can be found that the cyanobacteria of the sgRNA (SEQ ID NO. 1) of the present invention account for a higher percentage (75%) of the total colonies and compare with the sgRNA (SEQ ID NO. 2 and SEQ ID). The ratio of cyanobacteria to the total colonies of NO.3) is low (<8%, about 1%), as shown in Figure 6. This shows that the editing efficiency of the sgRNA of the present invention is high. In each plate, pick the blue colonies and culture them in LB liquid medium containing Cl-Amp, send the bacteria solution for sequencing, and the pMD-repeat plasmid repair sequencing maps are consistent, as shown in Figure 7.

In the blanc blue clone formation experiment, the replacement of the partial sequence of the lacZ gene of the pMD-19T plasmid destroyed the reading frame of β-gal. When transformed alone, β-gal could not be produced, showing white colonies; when the sgRNA corresponding to the target sequence was contained When the sequence of pCas9 plasmids is co-transformed, the Cas9 enzyme will cut the corresponding target sequence under the guidance of sgRNA to form a DNA double-strand break (DSB). The two repeated sequences undergo homologous recombination, which corrects the β-gal reading frame. Under the induction of X-gal and IPTG, blue colonies were formed. The proportion of blue colonies in all colonies reflects the editing activity of the sgRNA. If the sgRNA editing activity is low, the target sequence is reduced by pMD-19T plasmid, and the proportion of white colonies in the colonies will be high. It can be seen from the above that the sgRNA disclosed by the present invention has excellent editing efficiency and achieved unexpected technical effects.

Example two

The pSpCas9 (BB) -2A-GFP plasmid loaded with the sgRNA (SEQ ID NO. 1) of the present invention was constructed according to the conventional method described above. According to the above-mentioned conventional transfection system, pipette 0.8 μg of the successfully constructed pSpCas9 (BB) -2A-GFP plasmid and 2 μL of Lipo2000 into centrifuge tubes with 50 μL of pre-heated OMEM medium, mix gently, and let stand for 5 min; Mix the two tubes again, mix by shaking slightly, and let stand for 20min. Aspirate the cell culture medium, wash it with PBS solution, add 100 μL of mixed solution and 100 μL of pre-warmed OMEM medium to each well of a 24-well plate, place in a 5% CO ₂ incubator at 37 ° C for 4 h, and add 200 μL of complete medium. Incubate for 12 hours, aspirate the liquid from the culture plate, add 1 mL of complete medium, and put it into a 5% CO ₂ incubator for incubation at 37 ° C for 48 hours. Transfection of the experimental group (containing sgRNA of SEQ ID NO. 1) and the negative control group (containing no sgRNA) was performed simultaneously.

Preparation and culture of single cell clones

HeLa cells were transfected with plasmids in the experimental and control groups for 48 hours, washed with PBS, trypsinized, neutralized with the medium, centrifuged at 1000 rpm, 5 min, and resuspended the cells in 500 μL of PBS solution, filtered by a 40 μm cell strainer. The plasmid was labeled with GFP. Transfection into cells, under the action of excitation light, emits green fluorescence. A single cell was used for each well of the 96-well plate to determine whether the cells were fluorescent or not.

The sorted single cell clones are cultured in a medium containing 15% FBS, and the medium is exchanged and replenished for about 20 days. When the cell clones grow to the bottom of the 96-well small wells, the medium is removed by aspiration, and 100 μL of PBS solution is added gently. Wash, aspirate, and add 100 μL trypsin to digest in a 5% CO ₂ incubator. When the cells are observed to be round when they are about to fall off, aspirate the trypsin solution, add 200 μL culture medium, and mix by pipetting. Transfer to a 24-well culture plate. , Make up the volume of the culture medium to 1mL, and put it into a CO ₂ incubator and culture at 37 ° C. According to the method of cell passaging, transfer from a 24-well plate to a 6-well plate, and then transfer to a medium dish. A part of the cells were frozen and a part of the cells continued to be cultured.

Western Blot

Cell protein sample preparation : Aspirate and discard the treated intracellular culture medium, add pre-warmed PBS solution, wash and aspirate; add an appropriate amount of trypsin to digest in a 5% CO ₂ incubator until the cells are basically shed; add an appropriate amount of medium Neutralize the trypsin and gently mix until the cells are completely detached; transfer to a 1.5mL centrifuge tube, 1000rpm, 5min; aspirate the supernatant from the centrifuge tube, suck as much as possible, while the pellet is not sucked away; add a certain volume of cell lysis Mix well by pipetting and let stand on ice for about 10 minutes; add 2 × Sample Buffer of the same volume as the cell lysate, shake and mix; sonicate 3 times with 200W under ultrasonic cell pulverizer; insert the sample into the foam plate The sample was boiled in boiling water for 10 min. The sample was stored at -20 ° C.

SDS-PAGE gel electrophoresis : Fix the glass plate on a gel rack, configure the polyacrylamide separation gel of the required concentration, pour it between the gaps, and flatten it with isopropyl alcohol; after about 30 minutes, configure the gel with concentrated gel , Pour off isopropanol, wash with double distilled water 3 times, tilt and drain, then add concentrated gel, insert the comb; after the gel is solidified about 30min, remove the comb, install it in the electrophoresis tank, pour 1 × EB, Sample and protein marker; 120V electrophoresis, about 15min, wait for the sample to enter the separation gel, adjust to 150V, and continue electrophoresis; when the sample is close to the lower edge of the gel, stop electrophoresis.

Transfer film : PVDF membrane is activated by putting methanol into the pan, and 1 × TB of cold when it is poured into the dish; follow the order of sponge-two filter papers-PVDF membrane-separation gel-two filter papers-sponge, tighten the clips, transfer to In the transfer film tank, put ice cubes and pre-cooled 1 × TB; constant current transfer film, 260mA, 2h. After the film transfer is completed, place the PVDF film in 5% skimmed milk powder and shake gently for 30 minutes on a shaker.

Antibody incubation : Dilute the primary antibody with 1 × TBST at a certain ratio and store in ice. According to the Marker size band distribution, scissors cut out the protein band of interest; put the protein band of interest in the diluted primary antibody solution at 4 ° C overnight; remove the primary antibody, wash the membrane with 1 × TBST, and place on a shaker Gently shake for 10min, repeat once; dilute the secondary antibody with 1 × TBST at a certain ratio, put the PVDF membrane into the secondary antibody solution, and shake gently for 2h on the shaker; remove the secondary antibody, and wash the membrane twice with 1 × TBST. Shake gently on the shaker for 5 min each time, then wash twice with 1 × TBS, shake gently on the shaker for 5 min each time.

ECL chemiluminescence development : The A and B liquids in the ECL developer are mixed at a ratio of 1: 1. The cleaned film is blotted with toilet paper to dry the remaining liquid and placed in the mixed liquid, and incubated for 2 minutes. The film is placed on a tray and placed in a chemical In a luminous imager, develop and image.

The pSpCas9 (BB) -2A-GFP plasmid and the blank SpCas9 (BB) -2A-GFP plasmid loaded with sgRNA4 were transfected into HeLa cells by conventional transfection methods. After 48 hours, cells with green fluorescence were transfected into each well. The cells were sorted into a 96-well plate by flow cytometry, continued to grow, grown to a suitable density, and transferred to a 24-well plate, a 6-well plate, and a medium dish in order. Monoclonal cells sorted after transfection with pSpCas9 (BB) -2A-GFP plasmid loaded with sgRNA4 were recorded as the HeLa-DJ-1-KO group; blank SpCas9 (BB) -2A-GFP plasmids were sorted after transfection. The cloned cells were designated as HeLa-DJ-1-WT group.

In order to confirm whether DJ-1 was completely knocked out, proteins of HeLa-DJ-1-WT and 6 HeLa-DJ-1-KO cells were extracted and tested by Western Blot. Using β-actin as an internal reference protein, the results are shown in Figure 8. Compared with HeLa-DJ-1-WT, DJ-1 in HeLa-DJ-1-KO was not expressed at all, indicating that HeLa cells with the DJ-1 gene knocked out The plant was successfully constructed. In 6 monoclonal cell lines, the DJ-1 gene was completely knocked out, which also verified the reliability and excellent editing efficiency of the sgRNA results of the present invention.

Immunofluorescence experiment

Fix the cells . Remove the culture medium from the cell culture plate, wash and discard the PBS solution, add an appropriate amount of 4% paraformaldehyde, and let it stand for 5-10 minutes. Aspirate the paraformaldehyde solution and wash it in PBS solution 2 or 3 times. Dry the PBS solution, add 0.25% Triton X-100, and shake gently on the shaker for 5min. Add PBS solution and wash 2 ~ 3 times. Add PBST solution (0.1 ‰ Tween 20 in PBS solution), shake gently on the shaker for 1 h, and then wash with PBST solution 3 times. Aspirate the liquid in the well, add a certain amount of primary antibody diluted with PBST, and shake it gently on the shaker for 4h. Aspirate the liquid in the well and wash it with PBST 3 times. Aspirate the liquid in the well, add a certain amount of fluorescent secondary antibody diluted with PBST, wrap it with aluminum foil, and shake it gently on the shaker for 2h. Aspirate the liquid in the well and wash it with PBS 3 times. DAPI was diluted with a PBS solution at a certain ratio, added to the wells, stained for 5 minutes in the dark, washed 3 times with PBS, and then added an appropriate amount of PBS, and photographed under a microscope. In order to further determine the knockout effect of DJ-1 in monoclonal HeLa cells, DJ-1 expression was detected using fluorescent secondary antibody-labeled DJ-1 antibodies. HeLa-DJ-1-KO1, 2 and HeLa-DJ-1 were selected. -WT was used for immunofluorescence experiments. The immunofluorescence diagram is shown in Figure 9. Immunofluorescence showed that compared to HeLa-DJ-1-WT, DJ-1 in HeLa-DJ-1-KO1,2 was completely knocked out.

Genome PCR sequencing of DJ-1 knockout cells

The genomes of the cells showing complete knockout of DJ-1 in immunoblot experiments and immunofluorescence experiments were used to design the primers in the upstream and downstream sequences of sgRNA, and PCR was performed. The products were purified and ligated to T vectors. The TA cloning method was applied to DJ-1. The edited partial sequence of the gene was sequenced, compared with the original genome, and the success of DJ-1 knockout was detected at the DNA level.

HeLa-DJ-1-KO1 and KO2 were used to extract the cell genome through immunoblot experiments and immunofluorescence experiments. Primer PCR was designed, and the T vector was connected. Sequencing was performed near the target of sgRNA. The sequencing results were compared with the wild type genome. -DJ-1-KO1 caused a total of 1bp and 11bp deletions near the Cas9 restriction site; Hela-DJ-1-KO2 caused a 1bp insertion and 5bp deletion near the Cas9 restriction site. The above mutations can cause the DJ-1 reading frame to change, and the DJ-1 gene cannot be expressed. It was proved that DJ-1 knockout was successful at the DNA level, and a monoclonal HeLa cell line with DJ-1 gene knockout was obtained.

CCK-8 method was used to detect cell proliferation : cells in the experimental and control groups were washed with PBS, digested with trypsin, neutralized with medium, centrifuged, resuspended with fresh medium, and then diluted to a certain multiple to count the cells. Dilute to 30,000 cells per milliliter, mix well, add 100 μL per well to a 96-well plate, and place in a CO ₂ incubator for cultivation. After the cells were adhered to the cells for 12 hours, 10 μLCCK-8 reagent was added to each well, and the corresponding amount of cell culture medium and CCK-8 reagent but no cells were added as a negative control. Put it in a CO ₂ incubator and incubate for 1 h. Set the plate reader to 450 nm and measure the absorbance. The experimental group and the control group were set up with 5 duplicate wells. The above measured value is recorded as 12h, and the cells are added to 96-well plate culture for 24h, 48h, 72h, and 96h time points, and the above operations are performed respectively to obtain absorbance values at each time point. repeat three times.

To evaluate the effect of knockout of DJ-1 on the proliferation of human cervical cancer HeLa cells, HeLa-DJ-1-KO1,2 and HeLa-DJ-1-WT were selected for CCK-8 experiments. 3,000 cells per well, each cell is provided with five duplicate wells, and the absorbance of each well was measured at 12h, 24h, 48h, 72h, and 96h with a microplate reader. The experimental results are shown in Figure 10.

During the application of CRISPR / Cas9, the level of sgRNA specificity largely determines the efficiency of CRISPR / Cas9 editing. Among the sgRNAs for DJ-1 disclosed by the present invention with the best editing efficiency, it can be found that the editing efficiency of the three sgRNAs is very different, from <1% to 75%. After selecting sgRNA4, DJ-1 was used in a monoclonal cell line. Complete knockout of the gene indicates that the sgRNA of the optimal editing efficiency of the present invention is important.

Sequence Listing Free Content

Sequence Listing

<110> Zhangjiagang Institute of Industrial Technology, Soochow University

Suzhou University

<120> sgRNA screening for DJ-1 gene editing and its vector and application

<160> 21

<170> SIPOSequenceListing 1.0

<210> 1

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 1

acatcacggc tacactgtac tgg 23

<210> 2

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 2

agtacagtgt agccgtgatg tgg 23

<210> 3

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 3

ctgcacagat ggcggctatc agg 23

<210> 4

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 4

aaacagtaca gtgtagccgt gatg 24

<210> 5

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 5

aaaacatcac ggctacactg tact 24

<210> 6

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 6

aaacctgcac agatggcggc tatcg 25

<210> 7

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 7

aaaacgatag ccgccatctg tgcag 25

<210> 8

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 8

aaacacatca cggctacact gtacg 25

<210> 9

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 9

aaaacgtaca gtgtagccgt gatgt 25

<210> 10

<211> 34

<212> DNA

<213> Artificial Sequence

<400> 10

agcttcagta cagtgtagcc gtgatgtggg gtac 34

<210> 11

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 11

cccacatcac ggctacactg tactga 26

<210> 12

<211> 34

<212> DNA

<213> Artificial Sequence

<400> 12

agcttcctgc acagatggcg gctatcaggg gtac 34

<210> 13

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 13

ccctgatagc cgccatctgt gcagga 26

<210> 14

<211> 34

<212> DNA

<213> Artificial Sequence

<400> 14

agcttcacat cacggctaca ctgtactggg gtac 34

<210> 15

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 15

cccagtacag tgtagccgtg atgtga 26

<210> 16

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 16

caccgagtac agtgtagccg tgatg 25

<210> 17

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 17

aaaccatcac ggctacactg tact 24

<210> 18

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 18

caccgctgca cagatggcgg ctatc 25

<210> 19

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 19

aaacgatagc cgccatctgt gcagc 25

<210> 20

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 20

caccgcacat cacggctaca ctgtac 26

<210> 21

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 21

aaacgtacag tgtagccgtg atgtgc 26.

Claims (10)

A sgRNA directed to the DJ-1 gene, the sequence of the sgRNA directed to the DJ-1 gene is SEQ ID NO.1. The sgRNA for the DJ-1 gene according to claim 1, wherein the sgRNA for the DJ-1 gene has an editing efficiency of 75%. A drug targeting the DJ-1 gene includes the sgRNA shown in SEQ ID NO.1. The drug for the DJ-1 gene according to claim 3, wherein the drug for the DJ-1 gene further comprises a drug carrier. The drug for the DJ-1 gene according to claim 4, wherein the drug carrier is a polymer carrier or a cell carrier. A plasmid targeting the DJ-1 gene includes the sgRNA shown in SEQ ID NO.1. The plasmid for the DJ-1 gene according to claim 6, wherein the plasmid for the DJ-1 gene is a pSpCas9 (BB) -2A-GFP plasmid loaded with the sgRNA shown in SEQ ID NO.1. Application of the sgRNA shown in SEQ ID NO. 1 in the preparation of a drug directed against the DJ-1 gene. The application according to claim 8, wherein the sgRNA shown in SEQ ID NO. 1 has an editing efficiency of 75%. Application of the sgRNA shown in SEQ ID NO. 1 in the preparation of a drug for treating or relieving Parkinson's disease.