CN113215193B - Method for improving activity of gene knockout and base editing system by small molecule compound and application method thereof - Google Patents
Method for improving activity of gene knockout and base editing system by small molecule compound and application method thereof Download PDFInfo
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Abstract
The invention relates to a method for improving the activity of a gene knockout and base editing system by using a small molecular compound Rocilinostat or Nexturastat A, which is used for improving the activity of the gene knockout and base editing system in the gene editing system by adding the small molecular compound Rocilinostat or Nexturastat A to treat cells when the gene editing system is used for carrying out gene editing. So as to obtain higher gene editing efficiency, solve the problem of low activity of the accurate site-specific gene editing, and be beneficial to the research of screening clinical tumor therapeutic targets, drug development, pathogenic mutation correction, animal model preparation and the like.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to a method for improving the activity of a gene knockout and base editing system and an application method thereof.
Background
The gene editing technique refers to editing a DNA sequence directly using a nuclease, thereby achieving knockout, insertion/deletion, substitution, or the like of a specific DNA fragment. At present, the development and transformation of a gene editing tool are always the research focus of scientists in the field of life science in the 21 st century, and new gene editing tools are endlessly layered. The CRISPR/Cas9 editing system utilizes single-stranded guide RNA (sgRNA) to guide the Cas9 protein with nuclease cleavage activity to cleave specific targets, thus realizing gene knockout and site-directed modification.
The Cytosine Base Editor (CBE) is a novel gene editing tool capable of editing single base C to T, and is mainly formed by fusing cytosine deaminase with a catalytic function and CRISPR/Cas 9. The cytosine deaminase is divided into the following types according to sources: rat-derived rAPOBEC1 (rat apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like protein), lamprey-derived activation-induced cytosine deaminase (AID) analog (PmCDA 1) [6], human AID (hAID), human APOBEC3A. The CRISPR/Cas9 used in early base editing systems was dCas9 without nuclease activity, which did not cleave the target sequence, it is now common to cleave only one of the DNAs with single-stranded DNA nickase activity, nCas9 (D10A), which both Cas9 retain the ability to bind to single-stranded guide RNAs (sgrnas), and Cas9 and sgrnas together mediate the conversion of the bases by cytosine deaminase at a specific target sequence. Recently, a novel Adenine Base Editor (ABE) has been reported which can realize adenine a to guanine G.
When the CBE and the ABE system edit bases, DSB is not required to be generated, so that random insertion/Deletion (Indel) caused by non-homologous end-joining (NHEJ) repair in cells is avoided, safer gene editing is realized, and the method can be applied to the fields of editing human cells, constructing a gene mutation animal model, improving crops, treating mouse embryos and the like.
However, the existing gene knockout and base editing systems have low efficiency, and have the problem of low activity in accurate site-specific gene editing. Therefore, the gene editing activity of the gene knockout and base editing system is improved, the high-precision editing of the target gene is realized, and the application of the gene editing tool in the fields of gene therapy, animal breeding and the like can be expanded.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide a method for improving the activity of a gene knockout and base editing system by using a small molecular compound, namely Ricolinostat (Rocilinostat or ACY-1215) or Nexturelastat A, and an application method thereof.
To achieve the purpose, the invention provides a method for improving the activity of a gene knockout and base editing system by using a small molecular compound, namely, ricolinostat (Rocilinostat or ACY-1215) or Nexturastat A, wherein the small molecular compound, namely, ricolinostat (Rocilinostat or ACY-1215), or Nexturastat A is added to treat cells when the gene editing system is used for gene editing, and the Ricolinostat or Nexturastat A improves the activity of the gene knockout and base editing system in gene editing.
Preferably, the gene editing system consists of a corresponding expression vector of a gene knockout system or a base editing system, which is an expression vector of a Cytosine Base Editor (CBE) or an Adenine Base Editor (ABE), and an sgRNA expression vector targeting a genomic DNA target site, and the gene knockout system is SpCas9.
Preferably, spCas9 is used to target cleavage of the DNA duplex of the gene of interest, inducing non-homologous end joining (NHEJ) within the cell, thereby generating a frame shift in the open reading frame of the gene of interest.
Preferably, the base of interest C is converted to T or A to G using CBE or ABE.
Preferably, the gene editing system is a coding plasmid expressing CRISPR/Cas9 or single base editor proteins.
Preferably, a method for improving the activity of gene knockout and base editing systems by using the small molecule compound Ricolinostat (rocylinostat or ACY-1215) or neurostat a according to claim 1, comprising the steps of,
step 1, constructing a SpCas9, CBE or ABE expression vector; the CBE (comprising APOBEC1-nCas9-UGI sequence) is amplified by PCR and is connected with a eukaryotic expression vector px330 to obtain a px330-CBE expression vector.
Step 2, constructing sgRNA of a specific target genomic DNA target site; annealing the single-stranded DNA to obtain an sgRNA fragment, and connecting the sgRNA fragment to an expression vector pJET-U6, px330-CBE or px330 to obtain a corresponding expression vector containing the sgRNA;
step 3, transfecting HEK-293 cells, and transfecting SpCas9, ABE, CBE or CBE-NG expression vectors and sgRNA expression vectors introduced into the HEK-293 cells so as to cause gene editing to occur at a genomic DNA target site;
step 4, adding Ricolinostat (rocylinostat or ACY-1215) or Nexturelastat A after transfection;
step 5, extracting the transfected HEK-293 cell genome DNA;
and 6, performing high-throughput sequencing on the genomic DNA PCR product, performing PCR amplification by using a specific primer aiming at the target site sequence, constructing a high-throughput sequencing library by using the PCR product, and performing high-throughput sequencing.
Preferably, the genomic DNA target Site comprises human EMXI, FANCF, RNF, HBB, HEK-Site3, HEK-Site4, ABCA4, ABE-Site3, ABE-Site8, ABE-Site12, ABE-Site14, ABE-Site17, ABE-Site19 target sites.
Compared with the prior art, in the invention, the HDAC6 inhibitor Ricolinostat (rocylinostat or ACY-1215) or Nexturastat A is used for improving the activity of a gene knockout and base editing system, namely the activity of a SpCas9, ABE and CBE gene editor. The method has the advantages of obtaining higher gene editing efficiency, solving the problem of low activity of accurate site-specific gene editing, and being beneficial to the research of screening clinical tumor therapeutic targets, drug development, pathogenic mutation correction, animal model preparation and the like. Provides a new method for the efficient use of gene editors in mammalian cells.
Drawings
FIG. 1 is a schematic diagram of px330-CBE plasmid;
FIG. 2 is a schematic diagram of pdonor-BFP-IRES-PURO plasmid;
FIG. 3 is a schematic diagram of pdonor-ABCA4 plasmid;
FIG. 4 is a chart comparing the flow results data of a Ricolinostat (Rocilinostat or ACY-1215) or Nextureastratat A-aided CBE base editing system with CBE alone;
FIG. 5 is a high throughput sequencing result of the test of the activity of the Ricolinostat (Rocilinostat or ACY-1215) or Nexturelastat A-assisted gene knockout and base editing system in endogenous gene editing in example 4;
FIG. 6 is a schematic diagram of the repair of px330-CBE-NG in the ABCA4 gene and the repair results.
Detailed Description
In order to further describe the technical means and effects adopted by the present invention for achieving the intended purpose, the following detailed description will refer to the specific implementation, structure, characteristics and effects according to the present invention with reference to the accompanying drawings and preferred embodiments.
The invention relates to a method for improving the activity of a gene knockout and base editing system by using a small molecular compound Ricolinostat (rocylinostat or ACY-1215) or a Nexturastat A, which is shown in figures 1 to 6. When using the gene editing system for gene editing, small molecular compound Ricolinostat (rocylinostat or ACY-1215) or neurostat a is added to treat cells, and Ricolinostat (rocylinostat or ACY-1215) or neurostat a is added to improve the activity of the gene knockout and base editing system in the gene editing system.
The gene editing system consists of a corresponding expression vector of a gene knockout system or a base editing system and an sgRNA expression vector of a target genomic DNA target site. The base editing system comprises an expression vector of a Cytosine Base Editor (CBE) or an Adenine Base Editor (ABE). The gene knockout system is a SpCas9 expression vector. The SpCas9 is a CRISPR-Cas9 based gene knockout system. Wherein SpCas9 is used for targeted cleavage of the target gene DNA double strand, and non-homologous end joining (NHEJ) in cells is induced, so that the open reading frame of the target gene is shifted. Alternatively, the base of interest C is converted to T or A to G using CBE or ABE.
The gene editing system can be selected to express a coding plasmid for CRISPR/Cas9 or single base editor proteins. The HDAC6 inhibitor Ricolinostat (Rocilinostat or ACY-1215) or Nextureasatat A is used for improving the activity of a gene knockout and base editing system, so that higher gene editing efficiency is obtained, the problem of low activity of specific gene editing at an accurate site is solved, and the method is beneficial to the research of screening clinical tumor therapeutic targets, drug development, pathogenic mutation correction, animal model preparation and the like. Provides a new method for the efficient use of gene editors in mammalian cells.
The application method of the method for improving the activity of the gene knockout and base editing system by using the micromolecular compound Ricolinostat (Rocilinostat or ACY-1215) or Nexturastat A comprises the following steps of:
step 1, constructing a SpCas9, CBE or ABE expression vector;
step 2, constructing sgRNA of a specific target genomic DNA target site; annealing the single-stranded DNA to obtain an sgRNA fragment, and connecting the sgRNA fragment to an expression vector pJET-U6, px330-CBE or px330 to obtain a corresponding expression vector containing the sgRNA;
step 3, transfecting HEK-293 cells, and transfecting SpCas9, ABE, CBE or CBE-NG expression vectors and sgRNA expression vectors introduced into the HEK-293 cells so as to cause gene editing to occur at a genomic DNA target site;
step 4, adding Ricolinostat (rocylinostat or ACY-1215) or Nexturelastat A after transfection;
step 5, extracting the transfected HEK-293 cell genome DNA;
and 6, performing high-throughput sequencing on the genomic DNA PCR product, performing PCR amplification by using a specific primer aiming at the target site sequence, constructing a high-throughput sequencing library by using the PCR product, and performing high-throughput sequencing.
Wherein the target sites of the genomic DNA comprise target sites of EMXI, FANCF, RNF, HBB, HEK-Site3, HEK-Site4, ABCA4, ABE-Site3, ABE-Site8, ABE-Site12, ABE-Site14, ABE-Site17 and ABE-Site19 of human beings.
The following is set forth by way of specific examples:
a novel engineered genome editing tool of this example comprises a CBE protein expression vector or its transcriptional translation product, and HDAC6 inhibitors Ricolinostat (Rociliostat or ACY-1215) and Nextureasatat A, and the CBE partial sequence of px330-CBE expression vector is shown in SEQ ID NO.1.
The improvement is specifically as follows: SEQ ID NO.1CBE sequence
In the above embodiment, the specific experimental method includes the following steps:
1. construction of a CBE expression vector: the CBE (comprising APOBEC1-nCas9-UGI sequence) is amplified by PCR and is connected with a eukaryotic expression vector px330 to obtain a px330-CBE expression vector.
2. Construction of sgRNA expression vectors: annealing the single-stranded DNA to obtain an sgRNA fragment, and connecting the sgRNA fragment to an expression vector pJET-U6, px330-CBE or px330 to obtain a corresponding expression vector containing the sgRNA;
3. transfecting HEK-293 cells;
4. extracting genome DNA;
5. high throughput sequencing of genomic DNA PCR products;
example 1: construction of px330-CBE expression vectors
(1) The experimental materials include:
the primer is prepared by a conventional method, a restriction enzyme, a plasmid recombination kit, a plasmid extraction kit, a gel recovery kit, a high-fidelity DNA polymerase and a genome DNA extraction kit. Plasmid pZHW-PBE comprising the CBE sequence: SEQ ID NO.1. Plasmid px330, plasmid pCMV-ABEmax, plasmid pJET-U6. Plasmid pJET-U6 is a fragment of pJET1.2 (CloneJET PCR Cloning Kit, thermo Fisher Scientific) with U6 and a scafold fragment (see SEQ ID NO.2 for fragment sequence) attached to the backbone. Transfection reagent Turbofect and T4 DNA ligase.
Specifically, the sequence SEQ ID NO.2U6-scaffold
In this embodiment, the specific implementation steps include:
step 1: carrying out single enzyme digestion on plasmid px330 by using restriction endonuclease AgeI to obtain skeleton 1;
step 2: annealing the DNA fragment by using a primer (the sequence AvrII-F, avrII-R is shown in Table 1) to obtain a double-stranded DNA fragment, and connecting the double-stranded DNA fragment to the framework 1 to obtain px330-AvrII containing AvrII enzyme cutting sites;
step 3: the px330-AvrII and the pZHW-PBE are respectively digested with restriction endonucleases AvrII and SacI, and a 4300bp fragment generated by the digestion of the px330-AvrII and a 5200bp fragment generated by the digestion of the pZHW-PBE are connected to obtain an improved expression vector px330-CBE.
Step 4: construction of sgRNA expression vector: and (3) annealing primers (primer sequences are shown in table 1) targeting specific sites to obtain sgRNA fragments, and connecting the sgRNA fragments to an expression vector px330-CBE (BsaI single enzyme-digested skeleton) to obtain the px330-CBE-sgRNA expression vector.
The application method of the CBE base editor in this example is as follows:
step 1: constructing sgRNA aiming at a certain pathogenic gene DNA target point, and co-introducing the sgRNA and a CBE expression vector into HEK-293 cells to enable the sgRNA and the CBE expression vector to generate base editing at a target site;
step 2: constructing a BFP report system for evaluating CBE editing activity and efficiency, wherein the BFP gene sequence utilized by the BFP report system is SEQ ID NO.3;
step 3: constructing a CBE gene repair template ABCA4 cell line, wherein the utilized ABCA4 gene sequence is SEQ ID NO.4;
step 4: extracting the genome DNA of the HEK-293 cell after treatment, carrying out PCR amplification by using specific primers (the sequences of which are shown in Table 1) aiming at targets, constructing a high-throughput sequencing DNA library by using PCR products of the genome DNA, and carrying out high-throughput sequencing.
The improvement is specifically the SEQ ID NO.3BFP sequence
SEQ ID NO.4ABCA4 sequence
Example 2: establishment of BFP reporting system for CBE activity test
(1) A px330-AAVS1 vector was constructed comprising an sgRNA targeting the AAVS1 site (sequences AAVS1-sgRNA-F, AAVS1-sgRNA-R see Table 1). A vector (pdonor-BFP-IRES-PURO) expressing BFP and carrying an AAVS1 site homology arm was constructed, which contained AAVS1 homology arm, blue Fluorescent Protein (BFP), IRES and Puromycin (Puromycin) resistance genes.
(2) HEK-293 cell resuscitation: taking out the tube for freezing HEK-293 cells from liquid nitrogen, immediately putting the tube into a water bath kettle at 37 ℃ and slightly shaking the tube, and centrifuging the tube at 1000rpm for 3min after the liquid is completely melted (about 1-1.5 min); taking out, wiping and sterilizing with 75% alcohol, and placing on an ultra-clean workbench; removing the supernatant, adding 1ml of cell culture medium to resuspend the cells, transferring the cells to a 10cm culture dish containing 10ml of culture medium, and gently shaking the cells back and forth and left and right to uniformly distribute the cells in the culture dish; marking cell type and date, name of cultured person, etc., and cooling to 37deg.C, 5% CO 2 Culturing in an incubator, and performing subsequent operation after the cells adhere to the wall and grow up.
Preparation of complete medium: DMEM (high sugar) +10% fbs (fetal bovine serum) +1% pen/strep (penicillin 100U/ml, streptomycin 100 μg/ml)
(3) Digesting HEK-293 cells in the logarithmic growth phase by trypsin, inoculating the HEK-293 cells into a 6-well plate after cell counting for 24 hours, carrying out cell transfection, wherein the experimental group is co-transfected by pX330-AAVS1 and pdonor-BFP-IRES-PURO vectors, the negative control group is co-transfected by pX330 and pdonor-BFP-IRES-PURO vectors, and the rest conditions are the same as the experimental group; 24h after transfection, the liquid is changed, 48h later, the culture is transferred to a 10cm dish, and the culture is screened by using the antibiotic Puromycin with the concentration of 1 mug/ml; after 10d, the monoclonal cells were picked under a microscope to obtain a monoclonal HEK293-AAVS1-BFP cell line with integrated BFP-IRES-PURO gene.
(4) The monoclonal HEK293-AAVS1-BFP cells are cultured in an enlarged mode, and the cloned BFP genes are subjected to PCR detection (sequences of primers F1, R1, F2 and R2 are shown in table 1) to determine that the cloned BFP genes are knocked in, namely the HEK293-AAVS1-BFP cells.
Referring to fig. 4, example 3: determination of efficiency of a CBE base editing System assisted by Ricolinostat (Rocilinostat or ACY-1215) or Nextureastrastat A at a specific site and comparison with CBE alone
(1) sgRNA design: designing sgRNA at a specific site of BFP gene sequence, enabling a base sequence corresponding to amino acid (histidine) at position 66 of BFP protein to be located in an editing window of CBE, annealing a primer corresponding to the base sequence (the sequence BFP-sgRNA-F, BFP-sgRNA-R is shown in table 1), and introducing the annealed primer into a px330-CBE vector to obtain a px330-CBE-BFPsgRNA vector;
(2) HEK293-AAVS1-BFP cell count and inoculated in 24-well plate, cell density of each well was 0.8X10 5 Individual cells/wells;
(3) After 24h of cell growth, plasmid px330-CBE-BFPsgRNA (250 ng) was transfected with the transfection reagent TurboFect (Thermo Fisher, #R0531);
(4) HEK-293 cell transfection method: the plasmid to be transfected was mixed with 50. Mu.l DMEM containing 1.5. Mu.l of the transfection reagent Turbofect, and after blowing and mixing, left to stand at room temperature for 15min, 0.5ml of DMEM (Gibco, C11995500 CP) +10% FBS (Gibco, 16000 044) medium, and 0.8X10 were slowly added along the side walls of the dishes 5 Transfection was performed in 24-well plates of individual cells/well HEK293-AAVS1-BFP cells.
(5) After transfection, either Ricolinostat (Rocilinostat or ACY-1215) or Nexturelastat A was added at a final concentration of 2.5mM, and the control was added with a corresponding volume of DMSO.
(6) Fresh medium was changed 24h after transfection, and the corresponding concentration of drug was added, cells were collected 48h later, the ratio of GFP green fluorescent cells was examined by flow cytometry, and the promotion of CBE activity at specific sites by either Ricolinostat (rocylinostat or ACY-1215) or Nexturelastat A was judged according to the ratio change. As can be seen from the bar graph of the flow results in FIG. 4, small molecules of Ricolinostat (Rocilinostat or ACY-1215) or Nexturelastat A can enhance base editing activity.
Referring to fig. 5, example 4: testing of endogenous Gene editing Activity of the Ricolinostat (Rocilinostat or ACY-1215) or Nextureastratat A-assisted Gene knockout and base editing System
(1) sgRNA design: designing sgRNA according to the requirement of a subsequent experiment, annealing corresponding primers (corresponding sequences are shown in table 1, spCas9 is the same as the sgRNA sequence used by CBE) at different sites (EMXI, FANCF, RNF, HBB, HEK-Site3, HEK-Site 4) of an endogenous gene sequence, and introducing the annealed primers into a px330 or px330-CBE vector to obtain px330-sgRNA or px330-CBE-sgRNA vector; corresponding endogenous sites (ABE-Site 3, ABE-Site8, ABE-Site12, ABE-Site14, ABE-Site17 and ABE-Site 19) of ABE, annealing the primers corresponding to the corresponding primers (the corresponding sequences are shown in Table 1), and introducing the annealed primers into a pJET-U6 vector to obtain a pJET-U6-sgRNA vector;
(2) HEK293 cells were counted and seeded in 12-well plates with a cell density of 1.8X10 per well 5 Individual cells/wells;
(3) After 24h of cell growth, the plasmid px330-sgRNA or px330-CBE-sgRNA (750 ng) corresponding to each position is transfected by using a transfection reagent TurboFect (Thermo Fisher, #R0531); for the ABE system, plasmid pCMV-ABEmax (500 ng) or pJET-U6-sgRNA (250 ng) corresponding to each site was transfected with a transfection reagent TurboFect (Thermo Fisher, #R0531);
(4) HEK-293 cell transfection method: after mixing the plasmids to be transfected in proportion, 100. Mu.l DMEM containing 3. Mu.l of the transfection reagent Turbofect was mixed, and after blowing and mixing, the mixture was allowed to stand at room temperature for 15min, and 1ml of DMEM (Gibco, C11995500 CP) +10% FBS (Gibco, 16000 044) medium, and 1.8X10% medium were slowly added along the side walls of the dishes 5 Transfection was performed in 12-well plates of HEK293 cells per cell/well.
(5) After transfection, either Ricolinostat (Rocilinostat or ACY-1215) or Nexturelastat A was added at a final concentration of 2.5mM, and the control was added with a corresponding volume of DMSO.
(6) Fresh medium was changed 24h after transfection, and the corresponding concentration of drug was added and cells were collected 72h later.
(7) Extraction of cell genome DNA: a Vazyme company DNA extraction kit was used.
(8) High throughput sequencing of DNA: designing and amplifying primers (primer sequences are shown in table 1) of 12 endogenous gene loci EMXI, FANCF, RNF, HBB, HEK-Site3, HEK-Site4, ABE-Site3, ABE-Site8, ABE-Site12, ABE-Site14, ABE-Site17 and ABE-Site19, carrying out two rounds of high-fidelity DNA polymerase PCR, completing the library construction process of a second generation DNA sequencing technology, carrying out high throughput sequencing on library construction samples, and automatically analyzing the detailed variation information of multiple target loci of multiple samples by Hi-TOM online software. FIG. 5 is a high throughput sequencing result.
Example 5: establishment of HEK293-ABCA4 cell line
(1) Construction of recombinant vector pdonor-ABCA4:
the partial sequence of the mutant ABCA4 gene (shown in figure 4) is amplified by PCR and cloned to a vector pdonor-BFP-IRES-PURO to obtain the recombinant pdonor-ABCA4 vector.
(2) Construction of a monoclonal HEK293 cell line integrated with the mutant ABCA4 gene:
step 1: HEK293 cells were counted and seeded in 12-well plates with a cell density of 1.6X10 per well 5 Individual cells/wells;
step 2: cell transfection was performed 24h later, the experimental group was co-transfected with px330-AAVS1 and pdonor-ABCA4 vectors, the negative control group was co-transfected with px330 and pdonor-ABCA4 vectors, and the other conditions were the same as those of the experimental group;
step 3: changing the liquid 24h after transfection, and passaging to a 10cm dish after 48 h;
step 4: after 5d, the monoclonal cells were picked under a microscope to obtain a monoclonal HEK293-ABCA4 cell line with integrated mutant ABCA4 gene.
Referring to fig. 6, example 6: promotion of in situ repair of CBE-NG in Stargardt disease causing genes by Ricolinostat (Rocilinostat or ACY-1215) or Nexturastat A. (see FIG. 6a. This illustration; 6b. Monoclonal Sanger sequencing assay; 6c. High throughput sequencing statistics);
(1) Construction of CBE-NG expression vector: the primers (sequences NG-F, NG-R are shown in Table 1) were used to amplify a fragment of around 900bp on px330-SpCas9-NG (purchased from Addgene website, # 117919). The px330-CBE plasmid was double digested with the restriction enzymes PshAI and SacI to give a backbone of about 8200 bp. And (3) carrying out infusion connection on the amplified fragment and a framework by using ClonExpress II One Step Cloning Kit to obtain an improved expression vector px330-CBE-NG.
(2) sgRNA design: 2 sgRNA sequences (ABCA 4-site1 and ABCA4-site 2) are designed near the point mutation of the mutant ABCA4 gene sequence, and primers corresponding to the sequences (the sequences ABCA4-site1-sgRNA-F, ABCA-site 1-sgRNA-R, ABCA-site 2-sgRNA-F, ABCA-site 2-sgRNA-F are shown in Table 1) are annealed and introduced into a px330-CBE-NG vector to obtain a px330-CBE-NG-sgRNA vector;
(2) HEK293-ABCA4 cells were counted and seeded in 12-well plates with a cell density of 1.8X10 per well 5 Individual cells/wells;
(3) After 24h of cell growth, 2 site-corresponding plasmid px330-CBE-NG-sgRNA (750 NG) was transfected with the transfection reagent TurboFect (Thermo Fisher, #R0531);
(4) HEK-293 cell transfection method: after mixing the plasmids to be transfected in proportion, 100. Mu.l DMEM containing 3. Mu.l of the transfection reagent Turbofect was mixed, and after blowing and mixing, the mixture was allowed to stand at room temperature for 15min, and 1ml of DMEM (Gibco, C11995500 CP) +10% FBS (Gibco, 16000 044) medium, and 1.8X10% medium were slowly added along the side walls of the dishes 5 Transfection was performed in 12-well plates of HEK293 cells per cell/well.
(5) After transfection, either Ricolinostat (Rocilinostat or ACY-1215) or Nexturelastat A was added at a final concentration of 2.5mM, and the control was added with a corresponding volume of DMSO.
(6) Fresh medium was changed 24h after transfection, and the corresponding concentration of drug was added and cells were collected 72h later.
(7) Extraction of cell genome DNA: a Vazyme company DNA extraction kit was used.
(8) High throughput sequencing of DNA: according to the instruction provided by Nostoc source company, the primer (ABCA 4-On-F, ABCA4-On-R sequence is shown in table 1) is designed to amplify the ABCA4 gene locus, two rounds of high-fidelity DNA polymerase PCR are carried out, the library construction process of the second generation DNA sequencing technology can be completed, the library construction sample is subjected to high-throughput sequencing, and the detailed variation information of multiple target loci of multiple samples can be automatically resolved by Hi-TOM online software provided by the company.
(9) Meanwhile, PCR products of genomic DNA (the sequences of the primers ABCA4-F, ABCA4-R are shown in Table 1) were ligated to the pJET vector by T4 DNA ligase (NEB) and transformed, and about 20 single clones were picked the next day for Sanger sequencing.
TABLE 1 summary of primer sequences
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The present invention is not limited to the above embodiments, but is capable of modification and variation in detail, and other modifications and variations can be made by those skilled in the art without departing from the scope of the present invention.
Claims (6)
1. A method for improving the activity of a gene knockout and base editing system by a small molecule compound, which is characterized in that: the method is a non-therapeutic method, when a gene editing system is used for gene editing, small molecular compound Ricolinostat (rocylinostat or ACY-1215) or Nexturastat A is added to treat cells, and the small molecular compound Ricolinostat (rocylinostat or ACY-1215) or Nexturastat A can improve the activity of the gene knockout and base editing system in the gene editing system;
the gene editing system consists of a gene knockout system or a corresponding expression vector of the base editing system and an sgRNA expression vector of a target genomic DNA target site, wherein the base editing system is an expression vector of a Cytosine Base Editor (CBE) or an Adenine Base Editor (ABE), and the gene knockout system is a SpCas9 expression vector.
2. The method for improving activity of gene knockout and base editing system by using small molecule compound according to claim 1, wherein SpCas9 is used for targeting cleavage of target gene DNA double strand, inducing non-homologous end joining (NHEJ) in cell, and further generating frame shift of target gene open reading frame.
3. The method for improving activity of a gene knockout and base editing system according to claim 1, wherein the base C of interest is converted into T or A into G using CBE or ABE.
4. The method of increasing the activity of a gene knockout and base editing system of claim 1 wherein said gene editing system is a coding plasmid expressing CRISPR/Cas9 or single base editor protein.
5. A method of using the small molecule compound of claim 1 to increase the activity of a gene knockout and base editing system, comprising: comprises the steps of,
step 1, constructing a SpCas9, CBE or ABE expression vector;
step 2, constructing sgRNA of a specific targeted genomic DNA target site, annealing by using single-stranded DNA to obtain a sgRNA fragment, and connecting the sgRNA fragment into an expression vector pJET-U6, px330-CBE or px330 to obtain a corresponding expression vector containing the sgRNA;
step 3, transfecting HEK-293 cells, and transfecting and introducing SpCas9, ABE and CBE into the HEK-293 cells
Or CBE-NG expression vector and sgRNA expression vector, so that gene editing occurs at the target site of genome DNA;
step 4, adding Ricolinostat (rocylinostat or ACY-1215) or Nexturastat A after transfection;
step 5, extracting the transfected HEK-293 cell genome DNA;
and 6, performing high-throughput sequencing on the genomic DNA PCR product, performing PCR amplification by using a specific primer aiming at the target site sequence, constructing a high-throughput sequencing library by using the PCR product, and performing high-throughput sequencing.
6. The method for improving activity of a gene knockout and base editing system using a small molecule compound according to claim 5, wherein the method comprises the steps of: target sites of the genomic DNA include human EMXI, FANCF, RNF, HBB, HEK-Site3, HEK-Site4, ABCA4, ABE-Site3, ABE-Site8, ABE-Site12, ABE-Site14, ABE-Site17, and ABE-Site19 target sites.
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| WO2021020884A2 (en) * | 2019-07-31 | 2021-02-04 | 한양대학교 산학협력단 | Cytosine base-editing composition and use of same |
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