KR20170037028A

KR20170037028A - Primer for prohibitin2 gene remove using CRISPR/CAS9 system

Info

Publication number: KR20170037028A
Application number: KR1020150136075A
Authority: KR
Inventors: 신명근; 최현정
Original assignee: 전남대학교산학협력단
Priority date: 2015-09-25
Filing date: 2015-09-25
Publication date: 2017-04-04
Also published as: KR101745863B1

Abstract

The method of removing the protease 2 gene using the CRISPR / CAS9 system of the present invention and the primer used therein are specifically designed so that only the proteobin 2 gene can be selectively and precisely removed. Thus, the target gene Only the protease 2 gene is specifically and precisely removed and the removal efficiency is remarkably improved.

Description

(Primer for Prohibition 2 gene removal using CRISPR / CAS9 system using CRISPR / CAS9 system}

The present invention relates to a primer for the removal of the proBactin 2 gene using the CRISPR / CAS9 system.

Since the discovery of restriction enzymes that recognize and cut specific sequences of DNA in the 1970s, genetic engineering techniques have developed rapidly over time. However, the limitation of gene manipulation technology using restriction enzymes was clear. Specifically, the restriction enzyme has a short length of about 6 to 8 recognizable gene sequences, and there is a problem that only about 46 (4,096) ordered pairs can be distinguished. The CRISPR / CAS9 system, on the other hand, does not have this limitation and is theoretically applicable to higher life than human beings.

The CRISPR / CAS9 system is a genome editing method called a clustered regularly interspaced short palindromic repeat (CRISPR) gene scissors. It uses RNA (gRNA) that specifically binds to a specific nucleotide sequence and Cas9 nuclease . Using such a CRISPR / CAS9 system, it is possible to knock-out plasmids into cells or animals to inhibit the function of specific genes.

The CRISPR / CAS9 system was discovered by scientists only a few years ago, and is a very old way of organisms, such as bacteria, that keep themselves from bacteriophages. An organism has evolved over millions of years by cutting off the bacteriophage's DNA, sticking it to its own gene, and surviving through adaptive immunity, which has been studied in a simple and clear way to quickly edit the organism's DNA in the laboratory.

Specifically, the original CRISPR / CAS9 system stores a portion of the DNA of a virus previously infected by the bacteria in its own genome, then retrieves the information again when the virus invades, Protection mechanism. By using it in genome engineering, a primer that searches for the base sequence of a specific gene is made and paired with the enzyme Cas9, which is a cleavage enzyme, to cling to the target DNA sequence to cause DNA cleavage. Therefore, mutation occurs in DNA repair (repair) process.

Until the CRISPR / CAS9 system was discovered, DNA editing was possible only with a sophisticated laboratory, many years of experience, a huge amount of money, and a very small range that could be practically applied. However, the CRISPR / Resolved.

Accordingly, the CRISPR / CAS9 system is highly expected to be a tool for the development of stem cell and somatic cell mutations that cause genetic diseases or the development of therapeutic agents for cancer cells.

As a result, the CRISPR / CAS9 system is inexpensive and easy to use, making it a revolution in genetic research. Large-scale research institutes are gradually building up patents related to christopher, and are investing heavily in research funding in the public and private sectors as well as the Crisper project.

However, there are still many problems in applying the CRISPR / CAS9 system to the human body. For example, in order to effectively utilize the CRISPR / CAS9 system in the human body, safety is very important, specifically, the target gene must be definitely removed and genes other than the target gene should not be affected. This is because the CRISPR / CAS9 system is likely to cause mutations in non-target nucleotide sequences similar to the nucleotide sequence of the target gene, and this possibility should eliminate the risk or potential risk of unforeseen mutations or unanticipated fatal problems .

In particular, the technology using the CRISPR / CAS9 system for deliberately editing genes that are specific to target genes is complex and difficult. Therefore, in order to clarify the causative mechanism of human diseases including tumors, and to use the CRISPR / CAS9 system as a whole in the human body, it should be applied exclusively to the target gene.

Therefore, in order to identify the Prohibitin gene using the CRISPR / CAS9 system, and further to prevent the expression of the PROBITIN 2 gene without ultimately affecting other gene sequences, a primer and / Conditions using a CRISPR / CAS9 system suitable for the above initiator are required.

Korean Patent Publication No. KR2015-0091052A (2015.08.07)

In order to solve the above problems, it is an object of the present invention to provide a primer for the removal of the protease 2 gene using the CRISPR / CAS9 system in which only the protease 2 gene is specifically and specifically removed.

It is also an object of the present invention to provide a primer for the removal of a pro-bivin 2 gene using the CRISPR / CAS9 system, which specifically removes only the target gene, the proBytechin 2 gene, without affecting the non-target gene.

The present invention provides a primer for the removal of a prohibitin 2 gene using the CRISPR / CAS9 system, which is a forward primer of SEQ ID NO: 1.

The present invention provides a primer for the removal of the PROBITIN 2 gene using the CRISPR / CAS9 system, which is a reverse primer of SEQ ID NO: 2.

The present invention also provides a method for preparing a plasmid comprising the steps of: a) inserting a primer of SEQ ID NOs: 1 and 2 into a plasmid to which a CRISPR / CAS9 system is applied, b) transforming the plasmid into which the primer of step a) has been inserted into a competent cell ); And c) removing the protease 2 gene by using a CRISPR / CAS9 system comprising transfection of the transfected water-soluble cells of step b) Method.

In one example of the present invention, a target therapeutic agent, an assay kit or a gene removal kit including a primer for removing a protease 2 gene using the CRISPR / CAS9 system can be provided.

In one example of the present invention, a gene therapy method including a method for removing a protease 2 gene using the CRISPR / CAS9 system can be provided.

The primer for the removal of the PROBITIN 2 gene using the CRISPR / CAS9 system of the present invention is specially designed so that only the PROBITIN 2 gene is selectively and precisely removed, so that the target gene, PROBIVITIN 2 Only the gene is specifically and correctly removed, and the removal efficiency is remarkably improved.

FIG. 1 is a graph showing the results of electrophoretic bands and direct sequence analysis of the primers of SEQ ID NOS: 1 and 2 of the present invention according to Example 1 inserted into a plasmid and the plasmid. FIG.
FIG. 2 is a graph showing the relative intensity of the band of the proteobin 2 gene measured by Western blotting the transfected cells according to Example 1. FIG.
FIG. 3 is a graph showing the removal efficiency of the proteobin 2 gene (relative) by measuring Western blot of the cells to be transfected according to Example 1 (PHB2), Comparative Example 1 (PHB2-2) and Comparative Example 2 intensity of band.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

The drawings described in the present invention are provided by way of example so that a person skilled in the art can sufficiently convey the idea of the present invention. Therefore, the present invention is not limited to the illustrated drawings, but may be embodied in other forms, and the drawings may be exaggerated in order to clarify the spirit of the present invention.

In addition, unless otherwise defined, technical terms and scientific terms used in the present invention have the same meanings as those of ordinary skill in the art to which the present invention belongs. In the following description and the accompanying drawings, Description of known functions and configurations that may unnecessarily obscure the subject matter will be omitted.

Also, units of% used unclearly in the present invention means weight percent.

Until now, there have been various problems in applying the CRISPR / CAS9 system to the human body. For example, the CRISPR / CAS9 system is likely to mutate in a non-target base sequence similar to that of the target gene. In particular, But it is difficult to elaborate genetically.

Accordingly, in order to solve the above problems, the present invention provides a gene deletion method using a CRISPR / CAS9 system, that is, a target gene is selectively and selectively removed by knock-out, thereby effectively suppressing the expression of the target gene- (CRISPR / CAS9 system), and a primer used therefor. Therefore, by designing the primer specifically so that only the target gene is selectively and precisely removed, there is an effect that the efficiency of protease 2 gene removal is remarkably improved.

In one example of the present invention, a method for removing a protease 2 gene using a CRISPR / CAS9 system comprises the steps of: a) inserting a primer of SEQ ID NOs: 1 and 2 into a plasmid to which a CRISPR / CAS9 system is applied, b) Transforming the plasmid into a competent cell; and c) transfecting the transfected water-soluble cells of step b) into a target cell to transfect the proibytein 2 gene And removing it.

The forward primer of SEQ ID NO: 1 of the present invention is CACCGAAGGGTGCAGTACCTAAGC.

The reverse primer of SEQ ID NO: 2 of the present invention is AACCGCTTAGGTACTGCACCCTTC.

In one example of the present invention, the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 may contain one or two or more nucleotide sequences complementary to a part of the nucleotide sequence of proBy. In general molecular biotechnology, the use of a primer designed randomly or arbitrarily for the complementary base sequence of the gene of interest may not be a problem since it can be recognized and acted on. However, the probittin 2 gene using the CRISPR / CAS9 system In the case of the removal method, as described above, there is a problem that only the target gene, the proteobin 2 gene, is specifically and precisely removed. However, since the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 of the present invention are all applied to the CRISPR / CAS9 system, only the PROBITIN 2 gene can be specifically and accurately removed. In addition, since it does not affect the non-target gene, the mutation does not occur even in a similar non-target base sequence, for example, and the stability is remarkably improved.

As described above, the forward primer of SEQ ID NO: 1 of the present invention and the reverse primer of SEQ ID NO: 2 are applied to the method of removing the PROBITIN 2 gene using the CRISPR / CAS9 system, so that only the target gene, And the knock-out of the protein is inhibited by the proteobin 2 gene. Therefore, it can be positively used to identify the role of the property by the probitin 2 gene, and ultimately, it can be applied and applied to the development of a therapeutic agent for probitibin 2, for example, in a tumor. Specifically, a target therapeutic agent, an assay kit or a gene removal kit including a primer for removing a protease 2 gene using the CRISPR / CAS9 system of the present invention can be exemplified. In the CRISPR / CAS9 system of the present invention, And a gene therapy method (tumor treatment method and the like) including a tin 2 gene removal method can be exemplified.

In one example of the present invention, the plasmid of step a) is not limited as long as it is a plasmid to which the CRISPR / CAS9 system is applied. For example, pX330-U6-Chimeric_BB-CBh-hSpCas9 shown in FIG. 1 is exemplified . When the plasmid of FIG. 1 is used in the method for removing the protease 2 gene using the CRISPR / CAS9 system of the present invention, the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 of the present invention are inserted into the plasmid And the water-soluble cells containing the plasmid can be selectively isolated and effectively cultured in the step b), and further, the transfection efficiency can be further improved in the target cell in the step c) . However, this is a preferable example, but the present invention is not limited thereto.

In one embodiment of the present invention, the target cell in the step c) may include a probitin 2 gene sequence, a proline protein having a characteristic of a probitin 2 gene, , Or a biomodel such as a human colon cancer cell line (HCT-116 or the like) can be exemplified as long as it is used for research such as characterization and role identification as a living body model.

Hereinafter, the method for removing the protease 2 gene using the CRISPR / CAS9 system of the present invention will be described in detail, but the steps or processes described below are not limited to the present invention.

In one embodiment of the present invention, the step a) may include inserting the primers of SEQ ID NOS: 1 and 2 into a plasmid to which the CRISPR / CAS9 system is applied.

A) isolating a portion of the plasmid by cleaving a portion of the plasmid with a restriction enzyme, a2) dephosphorylating the isolated plasmid by truncating the fragment, and a3) isolating the plasmid from SEQ ID NOs: 1 and 2 Annealing the primers and ligation to the dephosphorylated plasmid.

In one embodiment of the present invention, the step b) may include transforming a plasmid into which the primer of the step a) has been inserted into a water-soluble cell. Thus, the plasmid into which the primers of the present invention are inserted is transformed into a water-soluble cell, thereby allowing transformation into the target cell in step c). In addition, the water-soluble cells containing the plasmid can be amplified through a process such as culture by the step b). In addition, water-soluble cells containing the plasmid can be obtained by additionally carrying out a step of selecting, using an infection strain, a step of transforming the water-soluble cells with a plasmid containing a gene sequence expressing the infectious immunological characteristic . In addition, since the protease 2 gene is removed by transfection of the plasmid into the target cell of the step c), the expression of the direct / indirect characteristic of the organism including the target cell or the target cell by the proteobin 2 .

As a specific example, it may further comprise the step of selecting the cells transformed with the plasmid between step b) and step c). For example, by using a plasmid in advance to contain a gene sequence that expresses the infectious immunity characteristic using an infectious strain such as Carbenicillin, the water-soluble cells containing the plasmid in which the inventive primers are inserted are selectively sorted And amplified.

In one embodiment of the present invention, the step c) may include a step of transfecting a plasmid into a target cell. Methods for transfecting a plasmid into which the primers of the present invention are inserted into a target cell can be used without limitation because there are various known methods.

As described above, in the case of using the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 and the PROBITIN 2 gene deletion method using the CRISPR / CAS9 system, only the PROBITIN 2 gene is specifically and selectively Can be finely removed. For example, it can be used to identify the role of pro-biotin 2 in a variety of human-derived cell lines and zebrafish biomodels, and ultimately the development of target therapeutics for target compounds such as tumors and cancers Target remedy, gene removal kit, gene analysis kit, etc.).

Hereinafter, the present invention will be described in detail with reference to Examples. However, the present invention is described in more detail with reference to the following Examples. However, the scope of the present invention is not limited by the following Examples.

The site of the plasmid (pX330-U6-Chimeric_BB-CBh-hSpCas9) to which the restriction enzyme was applied was cleaved using BbsI restriction enzyme and then isolated. Specifically, 2 μl of NEB buffer 2.1, 1 μl of BbsI, 5.1 μl of plasmid and 11.9 μl of distilled water (DW) were incubated at 37 ° C. for 2 hours, followed by electrophoresis (1 μl loading) PCR purification was performed using a kit.

Next, the isolated plasmid DNA (DNA of plasmid) was dephosphorylated. Specifically, 17 μl of DNA of plasmid, 1 μl of Shrimp alkaline phosphatase and 2 μl of SAP 10x buffer were reacted at 37 ° C for 5 minutes and then reacted at 65 ° C for 5 minutes.

Next, the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 were annealed. Specifically, 2 μl of each primer, 3 μl of H ⁺ buffer and 43 μl of distilled water were reacted at 100 ° C. for 3 minutes and then at 70 ° C. for 5 minutes.

Next, the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 annealed to the separated pX330-U6-Chimeric_BB-CBh-hSpCas9 were inserted (Ligation). Specifically, 1 μl of DNA of plasmid, 0.5 μl of each primer, 1.5 μl EB buffer, and 3 μl of Mighty Mix were reacted at room temperature for 12 minutes.

Next, the plasmid DNA into which the primer was inserted was transformed into a competent cell. Specifically, 6 μl of plasmid DNA with a primer inserted into 50 μl of competent cells of DHα was reacted on ice for 20 minutes, followed by reaction in a water bath of 42 ° C. for 90 seconds, followed by reaction on ice for 2 minutes . Subsequently, 150 μl of LB broth was dispensed, and then cultured in a 37 ° C water bath for 30 minutes. Subsequently, 150 μl of the cultured mixture was spread on a LB solid medium containing Carbenicillin, and then cultured in a 37 ° C. incubator for 12 hours. Then, 8 colonies were selected from the solid medium, and each of them was dissolved in LB broth (40 ml of LB broth, 40 l of cabenicillin) containing carbenicillin, and then cultured in a 37 ° C shaker incubator for 12 hours . Plasmid DNA was extracted from the cultured mixture, and the transformation was confirmed by electrophoresis and direct sequence analysis. Specifically, 1 μl of the NEB buffer, 1 μl of BSA (10 ×), 1 μl of XbaI (10 μM), and 1 μl of BSA (10 μl) were used to confirm the electrophoretic band at 7117 bp / 1392 bp after cleavage with restriction enzymes XbaI and SacII. 0.4 μl of Sac II, 1 μl of DNA of plasmid, and 6.2 μl of distilled water were reacted at 37 ° C. for 1 hour)

Next, transfection was performed using human-derived colorectal cancer cell line (SW480) as a target cell, and then it was confirmed by Western blot that the gene of the proteobin 2 gene was properly removed. The results are shown in FIGS. 2 and 3 .

Specifically, the above-described transfection method is as follows. The frozen SW480 cell stock was thawed in a 37 ° C water bath and added to 4 ml of DMEM medium (10% FBS (HyClone, GE Healthcare Life Science, Logan, USA), 1% P / S) And the supernatant was removed. 10 ml of DMEM medium was further added thereto and cultured in a 100 mm dish for 12 hours. Subsequently, the medium was removed, washed with PBS, added with 2 ml of 0.5% trypsin, allowed to react for 5 minutes in an incubator (37 ° C in a 5% CO ₂ incubator), and then added with 4 ml of DMEM medium The supernatant was removed by centrifugation at rpm for 5 minutes. Cells were counted by adding 7 ml of fresh DMEM medium, and SW480 cells at a concentration of 3 × 10 ⁶ / ml (confluence 80%) were dispensed in a 60 mm dish tissue culture plate. The cells were incubated in an incubator (37 ° C. in a 5% CO ₂ incubator) Lt; / RTI > for 12 hours. The medium of the cultured cells was replaced with 4 ml of DMEM medium supplemented with no P / S (antibiotics) and only FBS. Plasmid DNA, Lipofectamine 2000 (Invitrogen) and OPTI-MEM (Thermo Fisher Scientific) were prepared and 500 μl of OPTI-MEM medium was dispensed into each of the two EP tubes. 20 μl of Lipofectamine 2000 was added to No. 1 tube, After adding 8 ㎍ of plasmid DNA to the tube, mixture 1 was added to the mixture tube 2 times and mixed. The final mixture was reacted at room temperature for 25 minutes. After the reaction mixture was added dropwise to the tissue culture plate of the cultured cell replaced with 4 ml of the DMEM medium, the cells were cultured for 18 hours or more in an incubator (5% CO ₂ incubator at 37 ° C) Tin 2 gene was confirmed to be properly removed.

[Comparative Example 1]

(CACCGTTCTGACCTCCAGTGCCGCC) having the nucleotide sequence complementary to one part of the pro-biventin 2 base sequence, respectively, instead of the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 in Example 1 and the forward primer (AACCGGCGGCACTGGAGGTCAGAAC) was used as the reverse primer (AACCGGCGGCACTGGAGGTCAGAAC), and the results are shown in FIG.

[Comparative Example 2]

(CACCGCGCCGTGGCCTACGGTGTG) containing the nucleotide sequence complementary to one part of the pro-biventin 2 base sequence, respectively, instead of the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 in Example 1 and the forward primer of SEQ ID NO: (AACCCACACCTTAGGCCACGGCGC) was used as the reverse primer (AACCCACACCTTAGGCCACGGCGC), and the results are shown in FIG.

1 shows the results of electrophoretic bands and direct sequencing analysis of the primers of SEQ ID NOS: 1 and 2 of the present invention according to Example 1 inserted into a plasmid and the plasmid, and the results of the plasmid using the plasmid with the CRISPR / CAS9 system The reverse primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 of the present invention were properly inserted into the primer.

FIG. 2 shows the relative intensity of the band of the protobytin 2 gene measured by Western blotting of the transfected cells according to Example 1, and it can be confirmed that the proteobin 2 gene was effectively removed.

FIG. 3 is a graph showing the removal efficiency of the proteobin 2 gene (relative) by measuring Western blot of the cells to be transfected according to Example 1 (PHB2), Comparative Example 1 (PHB2-2) and Comparative Example 2 intensity of band. When using a common primer designed randomly or arbitrarily for the complementary base sequence of the gene of interest, the value of the relative intensity of band is about twice or more different from that of the primer of the present invention And it can be confirmed that only the target gene, that is, the probitin 2 gene, is specifically and precisely removed when a commonly used designed primer is used.

It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the appended claims and their equivalents.

<110> Chonnam National University <120> Primer for prohibitin2 gene remove using CRISPR / CAS9 System <130> P15070960828 <160> 6 <170> KoPatentin 3.0 <210> 1 <211> 24 <212> DNA <213> Unknown <220> <223> prohibitin2 <400> 1 caccgaaggg tgcagtacct aagc 24 <210> 2 <211> 24 <212> DNA <213> Unknown <220> <223> prohibitin2 <400> 2 aaccgcttag gtactgcacc cttc 24 <210> 3 <211> 25 <212> DNA <213> Unknown <220> <223> prohibitin2 <400> 3 caccgttctg acctccagtg ccgcc 25 <210> 4 <211> 25 <212> DNA <213> Unknown <220> <223> prohibitin2 <400> 4 aaccggcggc actggaggtc agaac 25 <210> 5 <211> 24 <212> DNA <213> Unknown <220> <223> prohibitin2 <400> 5 caccgcgccg tggcctacgg tgtg 24 <210> 6 <211> 24 <212> DNA <213> Unknown <220> <223> prohibitin2 <400> 6 aacccacacc gtaggccacg gcgc 24

Claims

A primer for the removal of the PROBITIN 2 gene using the CRISPR / CAS9 system, which is the forward primer of SEQ ID NO: 1.

A primer for the removal of the PROBITIN 2 gene using the CRISPR / CAS9 system, which is the reverse primer of SEQ ID NO: 2.

a) inserting the primers of SEQ ID NOS: 1 and 2 into a plasmid to which the CRISPR / CAS9 system is applied,
b) transforming the plasmid in which the primer of step a) is inserted into a water-soluble cell; and
c) transforming the transfected water-soluble cells of step b) into a target cell to remove the proteobin 2 gene,
/ RTI >< RTI ID = 0.0 > CRISPR / CAS9 < / RTI >

4. A target therapeutic agent comprising a prodrug of any one of claims 1 or 2.

A kit for removing a protease 2 gene comprising the primer of claim 1 or 2.

An assay kit comprising the primer of any one of claims 1 to 3.

A gene therapy method comprising a method for removing a protease 2 gene using the CRISPR / CAS9 system of claim 3.