GRNA TARGETING CTGF GENE AND USE THEREOF
20230212615 · 2023-07-06
Inventors
Cpc classification
C12N2310/20
CHEMISTRY; METALLURGY
C12N15/111
CHEMISTRY; METALLURGY
C12N9/22
CHEMISTRY; METALLURGY
C12N15/88
CHEMISTRY; METALLURGY
C12N2800/80
CHEMISTRY; METALLURGY
C12N15/11
CHEMISTRY; METALLURGY
C12N15/1136
CHEMISTRY; METALLURGY
C12N15/113
CHEMISTRY; METALLURGY
International classification
C12N15/90
CHEMISTRY; METALLURGY
C12N15/11
CHEMISTRY; METALLURGY
C12N9/22
CHEMISTRY; METALLURGY
Abstract
Provided are gRNA that can direct a Cas enzyme to target a CTGF gene and the use thereof, which belongs to the technical field of gene editing. The gRNA may direct the Cas enzyme to perform targeted cleavage on an SMAD binding site region of a CTGF gene promoter, or the gRNA may direct the Cas enzyme to perform targeted cleavage on a CTGF gene exon 2 region. The gRNA can reduce the overexpression of the human CTGF gene via a CRISPR-Cas gene editing system. The above-mentioned gRNA is used for preparing a drug for use against fibrotic diseases.
Claims
1. A gRNA, wherein the gRNA can guide a Cas enzyme to target SMAD binding site region of a CTGF gene promoter, or the gRNA can guide a Cas enzyme to target and cleave CTGF gene exon 2 region.
2. The gRNA according to claim 1, wherein sequence of SMAD binding site region is shown in SEQ ID NO: 38 or a reverse complementary sequence thereof, and sequence of the exon 2 region is shown in SEQ ID NO: 39 or a reverse complementary sequence thereof
3. The gRNA according to claim 2, wherein sequence of the SMAD binding site sequence is shown in SEQ ID NO: 40 or a reverse complementary sequence thereof
4. The gRNA according to claim 1, which comprises a targeting domain selected from the group consisting of: 1) a base sequence as shown in any one of SEQ ID NO: 1-SEQ ID NO: 32; or 2) an extended sequence having at least 40% sequence identity with any one of SEQ ID NO: 1-SEQ ID NO: 32.
5. The gRNA according to claim 4, wherein the base sequence is selected from any one of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20 or SEQ ID NO: 31.
6. A gRNA expression vector for targeting and editing CTGF gene, comprising a nucleotide sequence encoding the gRNA according to claim 1.
7. A CRISPR system for targeting and editing CTGF gene, comprising the gRNA according to claim 1.
8. The CRISPR system for targeting and editing CTGF gene of claim 7, which comprises a Cas enzyme.
9. The CRISPR system for targeting and editing CTGF gene of claim 8, wherein the Cas enzyme is Cas9, Cas12 or Cas13; the Cas9 includes but not limited to SpCas9, SaCas9, Nme2Cas9, Nme3Cas9, CjCas9, NmCas9, FnCas9, nCas9, and dCas9 molecules, and fusion proteins and mutants thereof; the Cas12 includes but not limited to Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas 12f, Cas12g, Cas12h, Cas12i, Cas12j, Cas12k, Cas121 and Cas12m molecules, and fusion proteins and mutants thereof
10. The CRISPR system for targeting and editing CTGF gene of claim 9, wherein the Cas enzyme is Cas9.
11. A composition for targeting and editing CTGF gene, comprising: a gRNA system and a Cas enzyme system, the gRNA system directly or indirectly comprising the gRNA according to claim 1, and the Cas enzyme system directly or indirectly comprising a Cas enzyme.
12. A composition for targeting and editing a CTGF gene, comprising: a gRNA system and a Cas enzyme system, the gRNA system directly or indirectly comprising a gRNA, and the Cas enzyme system directly or indirectly comprising a Cas enzyme; wherein the gRNA system is selected from the group consisting of: a gRNA, or a nucleotide encoding a gRNA; and the Cas enzyme system is selected from a Cas enzyme, or a nucleotide encoding a Cas enzyme; the gRNA is as defined in claim 1.
13. The composition for targeting and editing a CTGF gene according to claim 12, wherein the Cas enzyme is Cas9, Cas12 or Cas13; the Cas9 includes but not limited to SpCas9, SaCas9, Nme2Cas9, Nme3Cas9, CjCas9, NmCas9, FnCas9, nCas9, and dCas9 molecules, and fusion proteins and mutants thereof; the Cas12 includes but not limited to Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, Cas12i, Cas12j, Cas12k, Cas121 and Cas12m molecules, and fusion proteins and mutants thereof
14. The composition for targeting and editing a CTGF gene according to claim 13, the Cas enzyme is Cas9.
15. A liposome, comprising an active ingredient and a lipid component as a carrier, the active ingredient comprising a gRNA, a gRNA expression vector comprising a nucleotide sequence encoding a gRNA, a CRISPR system comprising a gRNA, or a composition comprising a gRNA system and a Cas enzyme system; the gRNA is as defined in claim 1.
16. A method for treating fibrotic diseases, comprising administrating a gRNA, a gRNA expression vector comprising a nucleotide sequence encoding a gRNA, a CRISPR system comprising a gRNA or a composition comprising a gRNA system and a Cas enzyme system, to a subject in need thereof; wherein the gRNA is as defined in claim 1.
17. The method according to claim 16, wherein the fibrotic disease is pulmonary fibrosis.
18. The method according to claim 17, wherein the medicament is administered by inhalation.
19. A method for treating fibrotic diseases, comprising administrating the liposome targeting CTGF gene of claim 15; preferably, the fibrotic disease is pulmonary fibrosis; more preferably, the subject is administrated through inhalation.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0072] In order to facilitate understanding of the present application, a more comprehensive description of the application will be described below with reference to the related drawings. Preferred examples of the present application are shown in the drawings. However, the present application may be implemented in many different forms and is not limited to the examples described herein. On the contrary, the purpose of examples these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.
[0073] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art. The terms used herein in the description of the present application are for the purpose of describing particular examples only and are not intended to limit the present application. As used herein, the term “and/or” means that any and all combinations of one or more of the relating listed items are included.
Definitions
[0074] The gRNA molecule described in the present application comprises a targeting domain complementary to CTGF gene sequence, and a fixed sequence domain (a backbone sequence). The gRNA molecules described in the present application may be chemically modified on any nucleotide.
[0075] The “Cas enzyme” described in the present application refers to CRISPR-related nucleases, including but not limited to CRISPR-related nuclease molecules or fusion proteins thereof, consisting of Type II, V, and VI nucleases. Wherein Type II nuclease is Cas9, Type V nuclease is Cas 12 and Type VI nuclease is Cas 13.
[0076] The “Cas9” described in the present application includes but not limited to SpCas9, SaCas9, Nme2Cas9, Nme3Cas9, CjCas9, NmCas9, FnCas9, nCas9, and dCas9 molecules, and fusion proteins and mutants thereof
[0077] The “Cas12” includes but not limited to Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas 12f, Cas12g, Cas12h, Cas12i, Cas12j, Cas12k, Cas121 and Cas12m molecules, and fusion proteins and mutants thereof. In one embodiment, Cas12 is AsCpf1.
[0078] The reagents and materials in the following embodiments are all commercially available sources unless otherwise specified; experimental methods are conventional experimental methods in the art unless otherwise specified.
EXAMPLE 1
[0079] CTGF gene promoter elements or exon 2 was edited by CRISPR gene editing methods.
1. Preparation of Vectors
[0080] 1) Determine the gRNA targeting domain (the same as the sequence of the target sequence):
[0081] According to the sequence adjacent to the SMAD binding site of the human CTGF gene promoter region and the exon 2 sequence, gRNAs with a targeting domain length of 17 nt-24 nt were designed. In addition, a gRNA (Responsive-sgRNAx) targeting the GTGTCAAGGGGTC (SEQ ID NO: 57) sequence of the TGFβ response element adjacent to the SMAD binding site was designed. Some of them are shown in Table 2.
TABLE-US-00002 TABLE 2 The designed gRNA targeting domains SEQ ID NO of targeting Targeting domain gRNA name domain sequences SMAD- SEQ ID NO: 1 GUGCCAGCUUUUUCAGA sgRNA1 SMAD- SEQ ID NO: 4 AGUGUGCCAGCUUUUUCAGA sgRNA2 SMAD- SEQ ID NO: 6 GGAGUGUGCCAGCUUUUUCAGA sgRNA3 SMAD- SEQ ID NO: 8 CUGGAGUGUGCCAGCUUUUUC sgRNA4 AGA SMAD- SEQ ID NO: 9 CCAGCUUUUUCAGACGG sgRNA5 SMAD- SEQ ID NO: 12 GUGCCAGCUUUUUCAGACGG sgRNA6 SMAD- SEQ ID NO: 14 GUGUGCCAGCUUUUUCAGACGG sgRNA7 SMAD- SEQ ID NO: 16 GAGUGUGCCAGCUUUUUCAGA sgRNA8 CGG Exon2-sgRNA1 SEQ ID NO: 18 CGUCUGCGCCAAGCAGCU Exon2-sgRNA2 SEQ ID NO: 20 CGCGUCUGCGCCAAGCAGCU Exon2-sgRNA3 SEQ ID NO: 31 CUGCCGCGUCUGCGCCAAG CAGC SMAD- SEQ ID NO: 9 CCAGCUUUUUCAGACGG sagRNAl SMAD- SEQ ID NO: 12 GUGCCAGCUUUUUCAGACGG sagRNA2 SMAD- SEQ ID NO: 14 GUGUGCCAGCUUUUUCAGACGG sagRNA3 SMAD- SEQ ID NO: 16 GAGUGUGCCAGCUUUUUCAGA sagRNA4 CGG Responsive- SEQ ID NO: 33 AGGAAUGCUGAGUGUCA sgRNA1 Responsive- SEQ ID NO: 34 CGGAGGAAUGCUGAGUGUCA sgRNA2 Responsive- SEQ ID NO: 35 CAGACGGAGGAAUGCUGAGU sgRNA3 GUCA
[0082] Among the gRNAs designed above, SMAD-sgRNAx, Exon2-sgRNAx, and Responsive-sgRNAx (x is the serial number) correspond to the subsequent construction of SpCas9 plasmid, and SMAD-sagRNAx (x is the serial number) corresponds to the subsequent construction of SaCas9 plasmid.
[0083] Sense strands and antisense strands (cacc was added to the 5′-end of the sense strand, and if the first nucleotide at the 5′-end of the sense strand is not guanine G, caccg was added to the 5′-end of the sense strand; aaac was added to the 5′-end of the antisense strand, if the first nucleotide of the 5′-end of the sense strand is not guanine G, C was added to the 3′-end of the antisense strand) corresponding to the DNA sequences of the gRNA targeting domain were synthesized using conventional methods.
[0084] The sense strands and antisense strands corresponding to the DNA sequences of the gRNA targeting sequences were mixed (each pair of sense strands and antisense strands [F/R strands] were tested separately), incubated at 95° C. in a PCR machine for 5 min. Next, the mixture was immediately taken out and incubated on ice for 5 min, and then annealed to form double-stranded DNA with sticky ends.
[0085] 2 μL of the annealed product was used and diluted 500-fold with deionized water.
2) T4 Ligation Reaction
[0086] The PX459 plasmid (containing SpCas9 and AmpR corresponding sequences) was digested using restriction endonuclease Bbs I, the enzyme digestion effect was detected by electrophoresis, and the PX459 vector enzyme digestion product was recovered. The linearized PX459 was recovered through gel cutting, and then ligated with the annealed double-stranded DNA. The reaction system is showing in the following table. The annealed product and the linearized backbone vector were incubated at 16° C. in a PCR machine for 1 hour to fulfill the ligation, and SpCas9 plasmid was obtained.
TABLE-US-00003 TABLE 3 Ligation reaction system Annealed product 2 μL (diluted 1000-fold) Linearized backbone vector 5 ng Solution I (Takara) 3 μL Deionized water Up to 6 μL
[0087] Using a similar method as described above, the pX601 plasmid (containing SaCas9 and AmpR corresponding sequences) was digested with restriction enzyme Bsa I, recovered and ligated to obtain SaCas9 plasmid.
[0088] For example, the plasmid sequences constructed by SMAD-sgRNA2 group and SMAD-sagRNA2 group are shown in SEQ ID NO: 36 and SEQ ID NO: 37, respectively.
2. Plasmid Transformation and Coating Ampicillin-resistant Solid Culture Plate
[0089] 1) In the super clean bench, all products in T4 ligation reaction were added into a tube (50 μL) comprising E. coli DH5a competent cells rapidly. Then, the tube was incubated on ice for 30 min.
[0090] 2) The competent cells were immersed and bathed in water at 42° C. for 90 sec, and then incubated on ice for 2 min.
[0091] 3) In the super clean bench, 400 μL of antibiotic-free LB medium was added to the bacterial solution. Then, the bacterial solution was put into a bacterial shaker and recovered at 200 rpm and 37° C. for 1 h. During recovery, the biochemical incubator was turned on and LB agar plates containing appropriate amount of ampicillin were dried in it.
[0092] 4) The bacterial solution was centrifuged at room temperature and 12,000 rpm for 1 min, then most of the supernatant was removed using a pipette, about 50 μL supernatant was left to resuspend the precipitate thoroughly.
[0093] 5) The bacterial solution was dropped on the edge of the ampicillin-containing LB agar plates and then streaked using a pipette tip. The plates were placed upside down in a biochemical incubator and incubated for 16-18 h.
[0094] 3. Positive clones were selected, and plasmids were extracted and sequenced after expansion culture.
[0095] 1) In the super clean bench, 7 single colonies were selected and added into 50 μL ampicillin-containing LB medium using 1-10 μL pipette tips separately. Then, the solution was pipetted several times to mix the bacteria with LB medium.
[0096] 2) 2 μL of the bacterial solution was added to the colony PCR reaction solution (shown in the table below), then mixed thoroughly. PCR reaction was performed after collecting the liquid at the bottom of the tube through instantaneous centrifugation. The remained bacterial solution was placed in a biochemical incubator for further culture. The primer sequence of PX459-R is GAGTGAAGCAGAACGTGGGG (SEQ ID NO: 41) and the primer sequence of pX601-R is GCTGGCA AGTGTAGCGGTCA (SEQ ID NO: 42).
TABLE-US-00004 TABLE 4 PCR reaction solution corresponding to SpCas9 plasmid strain 2 × Accurate Taq 10 μL; Master Mix (dye plus) U6 Promoter-F (10 μM) 0.25 μL; PX459-R (10 μM) 0.25 μL; Deionized water 7.5 μL
TABLE-US-00005 TABLE 5 PCR reaction solution corresponding to SaCas9 plasmid strain 2 × Accurate Taq 10 μL; Master Mix (dye plus) U6 Promoter-F (10 μM) 0.25 μL; pX601-R (10 μM) 0.25 μL; Deionized water 7.5 μL
[0097] 3) After PCR, 2 μL of PCR product was mixed with 1 μL of 6× Loading Buffer, then loaded into the sample wells of the agarose gel to run agarose gel electrophoresis.
[0098] 4) After finishing electrophoresis, the results were observed using the gel imaging system, and single clone in the electrophoresis strip with correct size and normal brightness were selected as positive clones.
[0099] 5) In super the clean bench, all the solutions of positive clones incubated in the biochemical incubator were added into 50 mL centrifuge tubes containing 5 mL ampicillin LB medium. After closing the lib, the centrifuge tubes were put into the bacterial shaker and tipsily fixed, cultured at 37° C. at 200 rpm for 16-18 hours.
[0100] 6) The bacterial solution was used for plasmid extraction, and the operation procedure was performed according to the instructions of the plasmid DNA extraction kit, and eluted with 50 μL Elution Buffer finally.
[0101] 7) According to the operation method of Qubit4 Fluorometer, the concentration of the plasmid was determined with the Qubit dsDNA BR Assay Kit.
[0102] 8) One positive clone per plasmid was picked, 5-10 μL of plasmid was provided for Sanger sequencing, and the sequencing primer was the universal U6-Promoter-F (ACGATACAAGGCTGTTAGAG (SEQ ID NO: 43)).
4. Transfecting Cells, Extracting Genomes, and Identifying Genotypes
[0103] HEK293T cells were transfected with Lipofectamine2000 and the transfection reagent consisted of Lipofectamine2000 and Cas9 plasmid. Cells were seeded in 24-well plates with 5 x10.sup.5 cells per well and 500 ng plasmid were added.
[0104] After 72 h transfection, cells were collected and digested, then genome was extracted.
[0105] Sequences that approximate 500 bp from upstream to downstream of the gRNA binding site were amplified using the following specific primers
TABLE-US-00006 CTGF-SMAD-PCR-F: (SEQ ID NO: 44) CTCAGCGGGGAAGAGTTGTT CTGF-SMAD-PCR-R: (SEQ ID NO: 45) TGCTGTTTGCCTCTTCAGCT CTGF-EXON2-PCR-F: (SEQ ID NO: 46) CTCAGTCCGAGCGGTTTCTT CTGF-EXON2-PCR-R: (SEQ ID NO: 47) ATGACCGCCGCCAGTATG Responsive-PCR-F: (SEQ ID NO: 48) CTCTTTGGAGAGTTTCAAGAGCC Responsive-PCR-R: (SEQ ID NO: 49) TCGAGCTGGAGGGTGGAGTC
[0106] Wherein, CTGF-SMAD-PCR-F and CTGF-SMAD-PCR-R were used to amplify fragments with approximate 500 bp from upstream to downstream of the SMAD binding site, and Responsive-PCR-F and Responsive-PCR-R were used to amplify fragments with approximate 250 bp from upstream to downstream of sequence GTGTCAAGGGGTC (SEQ ID NO: 57) of TGFβ response element, CTGF-EXON2-PCR-F and CTGF-EXON2-PCR-R were used to amplify fragments with approximate 500 bp from upstream to downstream of the cleavage site of exon 2 of CTGF gene.
[0107] The PCR reaction system was prepared as follows, with a total volume of 20 μL:
TABLE-US-00007 2 × Accurate Taq 10 μL Master Mix (dye plus) Primer F (10 μM) 0.25 μL Primer R (10 μM) 0.25 μL Deionized water 8.5 μL Genome DNA templete (50 ng) 1 μL
[0108] PCR products were detected by 1% agarose electrophoresis, and partial test results were shown in
[0109] Then, the PCR products were recovered with kits and subjected to Sanger sequencing, and the experiments were repeated three times for each plasmid.
[0110] The sequencing results were imported into TIDE analysis website (https://ice.synthego.com/#/) to obtain gene editing efficiency, and the results were shown in the table below and
TABLE-US-00008 TABLE 6 Gene editing efficiency of each group Targeting Insertion/ domain Cas Deletion gRNA name sequences enzymes Indel(%) SMAD-sgRNA1 SEQ ID NO: 1 SpCas9 66 SMAD-sgRNA2 SEQ ID NO: 4 SpCas9 57 SMAD-sgRNA3 SEQ ID NO: 6 SpCas9 78 SMAD-sgRNA4 SEQ ID NO: 8 SpCas9 89 SMAD-sgRNA5 SEQ ID NO: 9 SpCas9 66 SMAD-sgRNA6 SEQ ID NO: 12 SpCas9 91 SMAD-sgRNA7 SEQ ID NO: 14 SpCas9 71 SMAD-sgRNA8 SEQ ID NO: 16 SpCas9 79 Exon2-sgRNA1 SEQ ID NO: 18 SpCas9 66 Exon2-sgRNA2 SEQ ID NO: 20 SpCas9 58 Exon2-sgRNA3 SEQ ID NO: 31 SpCas9 79 SMAD-sagRNA1 SEQ ID NO: 9 SaCas9 74 SMAD-sagRNA2 SEQ ID NO: 12 SaCas9 78 SMAD-sagRNA3 SEQ ID NO: 14 SaCas9 84 SMAD-sagRNA4 SEQ ID NO: 16 SaCas9 69 Responsive-sgRNA1 SEQ ID NO: 33 SpCas9 50 Responsive-sgRNA2 SEQ ID NO: 34 SpCas9 79 Responsive-sgRNA3 SEQ ID NO: 35 SpCas9 62
[0111] From the above results, it can be seen that the editing efficiency of all gRNA designed elaborately by present inventor can meet requirements, when targeting the SMAD binding site, sequence GTGTCAAGGGGTC (SEQ ID NO: 57) of TGFβ response element, and sequence GCTGCCGCGTCTGCGCCAAGCAGCT (SEQ ID NO: 58) of exon 2.
EXAMPLE 2
[0112] Gene editing on pulmonary fibrosis cell models.
1. Experimental Materials
[0113] The Nucleic Acid Purification Kit and 2× Accurate Taq master Mix, Reverse Transcription Kit, and 2X SYBR® Green Pro Taq HS Premix were purchased from Accurate Biotechnology (Hunan) Co., Ltd., OPTI-MEM and Lipofectamine 3000 transfection reagents were purchased from Thermo company, and TGF-β1, SpCas9 protein, and SaCas9 protein were purchased from Novoprotein. The primers and related RNAs used in PCR and sequencing were synthesized by reagent companies. A549 cells are commercially available.
2. Experimental Methods
2.1 Experimental Grouping
[0114] The groups are as follows:
[0115] The A549 group: blank control without any treatment;
[0116] The A549 TGF-β1 group: cells were treated with TGF-β1 only, without transfecting with RNP.
[0117] SMAD-sgRNAx group (x is the serial number): wherein the gRNA targeting domain is the same as that of Example 1, the complete sequence of the gRNA consists of targeting domain-backbone sequence, the backbone sequence is GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA ACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 50).
[0118] SMAD-sagRNAx group (x is the serial number): wherein the corresponding gRNA targeting domain is same as that of Example 1, the complete sequence of gRNA consists of targeting domain-backbone sequence, the backbone sequence consists of GUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCG UGUUUAUCUCGUCAACUUGUUGGCGAGAUUUUU (SEQ ID NO: 51).
[0119] Responsive-sgRNAx group (x is the serial number): the gRNA in this group targets sequence GTGTCAAGGGGTC (SEQ ID NO: 57) of the TGFβ response element adjacent to the SMAD binding site, the gRNA targeting domain is shown in the following table, and the complete sequence of gRNA consist of targeting domain-backbone sequence, the backbone sequence is GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA ACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 50).
[0120] The negative control group: the targeting sequence of gRNA is sequence TGTATGTCAGTGGACAGAAC at a distance of about 200 nt to the SMAD binding site of CTGF gene, and the complete sequence of gRNA is UGUAUGUCAGUGGACAGAACGUUUUAGAGCUAGAAAUAGCAAGUUAAA AUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUU (SEQ ID NO: 52).
[0121] In fact, the gRNAs used in each group of SMAD-sgRNAx, SMAD-sagRNAx, and Responsive-sgRNAx are the same as the gRNA sequence transcribed by the plasmids in Example 1.
2.2 Experimental Steps
2.2.1. Cell Treatment
[0122] A549 cells were seeded into 24-well plates at 1×10.sup.5/well for one day before transfection.
2.2.2. RNP Transfection
(1) RNP Transfection of SpCas9
[0123] The gRNA molecules in groups of SMAD-sgRNAx, Responsive-sgRNAx, and negative control were chemically synthesized.
[0124] Transfection complex A1 preparation: 6 pmol SpCas9 protein (Novoprotein, E365-01A) was added into 25 μL of OPTI-MEM medium (Thermo, 2120588), then 12 pmol gRNA was added to each group separately. The medium was mixed gently and placed at room temperature for 20 min to form RNP complex;
[0125] Transfection complex B1 preparation: 3 μL of Lipofectamine 3000 (Thermo, L3000-15) transfection reagent was added into 25 μL of OPTI-MEM medium. The medium was mixed gently and placed at room temperature for 5 min;
[0126] Transfection Complex Al was added into Transfection Complex B1 and gently mixed, then placed at room temperature for 15 min, followed by adding the mixture to the cells and culturing the cells for another 24 h.
(2) RNP Transfection of SaCas9
[0127] The gRNA molecules inSMAD-sagRNAx group were chemically synthesized.
[0128] Transfection complex A2 preparation: 4 pmol SaCas9 protein (Novoprotein, E372-01A) and 8 pmol gRNA were added into μL of OPTI-MEM medium. The medium was mixed gently and placed at room temperature for 20 min to form RNP complex;
[0129] Transfection complex B2 preparation: 2 μL of Lipofectamine 3000 transfection reagent was added into 25 μL of OPTI-MEM medium. The medium was mixed gently and placed at room temperature for 5 min;
[0130] Transfection Complex A2 was added to Transfection Complex B2 and gently mixed, then placed at room temperature for 15 min, followed by adding the mixture to the cells and culturing the cells for another 24 h.
[0131] 2.2.3. TGF-β1 (Novoprotein, P01137) was added to the cells to a final concentration of 10 ng/mL and the incubation was continued for 48 h.
[0132] 2.2.4. Cell samples were collected to extract RNA using SteadyPure Universal RNA Extraction Kit (Accurate Biology, AG21017).
[0133] 2.2.5. For RNA samples, gDNA was removed and reverse transcription reactions were performed using Evo M-MLV RT Kit with gDNA Clean for qPCR II (Accurate Biology, AG21017).
[0134] 2.2.6. Relative quantitative QPCR was used to detect changes in the relative expression of CTGF mRNA with GAPDH as the reference gene. The primer design and QPCR reaction system are as follows:
[0135] Primer Design:
TABLE-US-00009 hCTGF-QPCR-F: (SEQ ID NO: 53) GCGTGTGCACCGCCAAAGAT hCTGF-QPCR-R: (SEQ ID NO: 54) AACGTCCATGCTGCACAGGG hGAPDH-QPCR-F: (SEQ ID NO: 55) GGAAACTGTGGCGTGATGGC hGAPDH-QPCR-R: (SEQ ID NO: 56) GCTTCACCACCTTCTTGATGTC
[0136] QPCR reaction system:
TABLE-US-00010 2X SYBR ® Green 10 μL Pro Taq HS Premix Primer F (10 μM) 0.4 μL Primer R (10 μM) 0.4 μL cDNA Template .sub. 2 μL RNase free water up to 10 μL
2.2.7. Each Group Repeated the Experiment for 3 Times.
3. Experimental Results
[0137] The results were analysed using the 2-ΔΔCT method, and the results were shown in the table below and
TABLE-US-00011 TABLE 7 hCTGF mRNA relative expression hCTGF hCTGF mRNA mRNA relative relative groups expression group expression A549 100% Negative 3062% control A549 TGF-β1 2794% SMAD-sgRNA1 922% * SMAD-sagRNA1 598% * SMAD-sgRNA2 296% * SMAD-sagRNA2 276% * SMAD-sgRNA3 697% * SMAD-sagRNA3 945% * SMAD-sgRNA4 1139% * SMAD-sagRNA4 608% * SMAD-sgRNA5 840% * Responsive-sgRNA1 2066% SMAD-sgRNA6 753% * Responsive-sgRNA2 1689% SMAD-sgRNA7 992% * Responsive-sgRNA3 2313% SMAD-sgRNA8 565% * Note: * indicates statistically significant difference (P < 0.05) compared to the Responsive-sgRNAx group.
[0138] The above experimental results proved that the modeling is successful. The inventor was surprised to find that the SMAD-sagRNAx and SMAD-sgRNAx were more effective at reducing hCTGF mRNA expression than the Responsive-sgRNAx group.
[0139] The above experimental results showed that gRNA or CRISPR-Cas system that target and edit SMAD binding sites can reduce the overexpression of CTGF gene more effectively than targeting and editing sequence GTGTCAAGGGGTC (SEQ ID NO: 57) of TGFβ response elements.
[0140] In addition, edition of SMAD binding site by using SaCas9 and SpCas9 with the gRNA of the present application separately significantly reduced the expression of hCTGF mRNA, both of them had good effects, although SaCas9 and SpCas9 used different Cas enzymes and involved different PAM sequences.
[0141] The technical features of the example described above may be combined arbitrarily. To make the description concise, not all of possible combinations of the technical features in the above examples were described, however, as long as there is no contradiction in the combination of these technical features, such combinations should be considered to fall into the scope of the present description.
[0142] The examples described above only show several examples of the present application, and the description thereof is more specific and detailed, but it cannot be understood as a limitation to the scope of the patent application. It should be noted that for an ordinary person skilled in the art, without departing from the concept of the present application, certain variations and improvements may also be made, which should all fall into the protection scope of the present application. Therefore, the scope of protection of the present application shall be defined by the attached claims.