WO2018151339A1 - Method for detecting target gene, using dcas9/grna complex and fluorescence marker - Google Patents

Abstract

The present invention relates to a method for detecting a target gene, the method comprising: a first step of reacting a complex composed of deactivated Cas9 (dCas9) and a guide RNA specifically binding to a target gene with a sample including a target gene; a second step of separating the reactants; and a third step of treating with a fluorescence marker. A method for detecting a target gene according to the present invention allows the detection of a target gene at high sensitivity even without performing PCR and can be useful for detecting a target gene rapidly and accurately, without a separate gene isolation step.

Description

Target gene detection method using DCAS9 / GRNA complex and fluorescent marker

The present invention relates to a method of detecting a target gene using a dCas9 / gRNA complex and a fluorescent marker, and more particularly, to a target gene and a complex comprising an inactivated Cas9 (dCas9) and a guide RNA that specifically binds to a target gene. A first step of reacting the sample containing; A second step of separating the complex from the reactant; And a third step of processing the fluorescent labeling factor.

Until now, a method of detecting a gene has been reported based on a PCR method. Simple-direct tube RT-PCR (SDT / RT-PCR) method using direct PCR and IC / RT-PCR (Immunocapture RT-PCR) combining immunological methods to increase sensitivity and eliminate factors that inhibit PCR reaction Has been developed and used for gene detection.

In particular, it is necessary to quickly and accurately diagnose whether a pathogen is infected in order to cope early with disease caused by pathogen infection and to prevent disease progression and spread. If the pathogen can be diagnosed during the incubation period before the symptoms appear after infection, it can effectively prevent the spread of infectious diseases and prevent major damage.

However, the methods developed to date are difficult to prepare a sample which must extract a target gene to detect a gene to be identified, and it takes a lot of time when culturing cells. Furthermore, there is a problem that the abuse of drugs is continuously performed due to the lack of rapid diagnosis and lack of accuracy. Therefore, there is still a need for research on rapid and accurate gene detection.

Accordingly, the present inventors have made diligent efforts to develop a fast and accurate gene detection method. As a result, unlike the conventional gene diagnosis method in which PCR is essential or separates and analyzes only genes, the present inventors perform a separate gene separation step and PCR process. It was confirmed that the detection of the target gene with high sensitivity without having to complete the present invention.

[Preceding technical literature]

[Patent Documents]

Domestic Publication No. 10-2013-0094498

One object of the present invention is a first step of reacting a sample comprising a target gene and a complex consisting of a guide RNA that specifically binds to the target gene and inactivated Cas9 (dCas9); A second step of separating the complex from the reactant; And it provides a method for detecting a target gene, comprising a third step of processing the fluorescent marker.

Another object of the invention is a first step of lysing a cell comprising a target gene; A second step of reacting the lysate with a complex consisting of inactivated Cas9 (dCas9) and a guide RNA that specifically binds to a target gene; A third step of separating the complex from the reactant; And it provides a method for detecting a target gene, comprising a fourth step of processing the fluorescent marker.

As one aspect for solving the above problems, the present invention comprises a first step of reacting a sample comprising a target gene and a complex consisting of a guide RNA that specifically binds to a target gene and inactivated Cas9 (dCas9); A second step of separating the complex from the reactant; And a third step of processing the fluorescent labeling factor.

In detecting the target gene, the inventors of the present invention insist that the DNA amplification process is essential when using a fluorescent marker and a complex composed of inactivated Cas9 (dCas9) and guide RNA specifically binding to the target gene. In addition to simplifying the complex procedure of the molecular diagnostic method, it was confirmed that the shortcomings of the low sensitivity immunodiagnosis can be overcome, thereby completing a rapid and sensitive target gene specific detection method.

The method for detecting a target gene of the present invention includes a first step of reacting a sample comprising a target gene with a complex consisting of inactivated Cas9 (dCas9) and guide RNA specifically binding to the target gene.

The first step is a step of reacting the complex with a sample containing two or more genes including a target gene, the conjugate complexed with the target gene through the reaction, genes other than the unreacted target gene and the reaction It is possible to provide a reactant comprising a complex that is not.

In the present invention, the complex consisting of the inactivated Cas9 (dCas9) and the guide RNA specifically binding to the target gene may be formed before performing the method of detecting the target gene, but is not limited thereto. In practice, dCas9 and guide RNA may be formed by reacting the sample sequentially or together.

As used herein, the term "sample" refers to any sample that contains a target gene.

As used herein, the term “guide RNA” is an RNA including a sequence that specifically binds to a target gene. The guide RNA of the present invention may form a complex with a Cas9 protein. The guide RNA may be composed of crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA).

crRNA can bind to a target gene.

tracrRNA may bind to crRNA and change the structure of the dCas9 protein.

Specifically, the guide RNA in the present invention may be sgRNA connected in one strand while maintaining the role of crRNA and tracrRNA.

In the present invention, the term “specific binding” can be used interchangeably with hybridization.

Specific binding of the guide RNA to the target gene may mean that the guide RNA of a sequence complementary to the target gene hybridizes with the single stranded target sequence of the target gene to form a double-stranded molecule (hybrid).

The sequence complementary to the target gene of the guide RNA may be hybridized with a portion of the target gene, and the complementary sequence is at least 90%, specifically at least 95%, more specifically 100% complementary to the portion of the target gene. May be a sequence.

The term “inactivated Cas9” in the present invention is a Cas9 nuclease protein in which nuclease function is inactivated, and may also be referred to as dCas9. Preparation of the inactivated Cas9 protein may be prepared according to conventional methods of inactivating the activity of nucleases, but is not limited thereto.

The Cas9 protein and its genetic information can be obtained from a known database such as GenBank of the National Center for Biotechnology Information (NCMBI).

The method for detecting a target gene of the present invention includes a second step of separating the complex from the reactant.

The second step is the step of separating the complex from the reactant of the sample and the complex containing the dCas9 protein, the dCas9 protein may include an affinity tag (tag) used for separation or purification according to the purpose.

The affinity tag is His tag, Flag tag, S tag, GST (Glutathione S-transferase) tag, MBP (Maltose binding protein) tag, CMP (chitin binding protein) tag, Avi tag, calmodulin tag, Polyglutamate Tag, E Tag, HA Tag, myc Tag, SBP Tag, Softtag 1, Softag 3, Strap (strep) Tag, TC Tag, Xpress Tag, BCCP (biotin carboxyl carrier pretein tag, and GFP (green fluorescent protein) tag. Specifically, the dCas9 protein of the present invention may comprise His tag.

In the second step, the composite may be separated by using magnetic beads binding to the tag, and the magnetic beads may be Ni-NTA magnetic beads, but are not limited thereto.

The method for detecting a target gene of the present invention includes a third step of treating a fluorescent marker on the isolated complex.

The third step is a step of processing the fluorescent markers in the isolated complex, and only the complexes coupled to the target gene among the complexes can specifically detect the target gene by having a fluorescent signal.

The complex associated with the target gene has a double-stranded DNA, unlike the complex not bound to the target gene, and the complex associated with the target gene can be identified as a fluorescent marker that can specifically bind to the double-stranded DNA. .

As used herein, the term “fluorescent labeling factor” is a fluorescent material used to color nucleic acids and has a property of specifically binding to double stranded DNA. In the present invention, the fluorescent marker may specifically bind to the double-stranded DNA of the target gene bound to the complex.

The fluorescent labeling factor may be a fluorescent labeling factor used to detect double stranded DNA.

Specifically, in the group including SYBR green I, SYBR green II, SYBR gold, Oxazole yellow, YOYO and Thiazole orange It may be selected, but is not limited thereto.

In one embodiment of the present invention, after reacting a complex of sgRNA and dCas9 with a sample containing a variety of genes, including the target gene, after separating the complex with magnetic beads using the His tag of the dCas9 protein SYBR green on the eluted sample I was treated. As a result of observing the fluorescence signal, it was confirmed that a particularly strong fluorescence signal was detected in the target gene, and it was found that the target gene could be specifically detected through the above method (FIG. 3).

In another aspect, the present invention, the first step of lysing a biological sample containing a target gene; A second step of reacting the lysate with a complex consisting of inactivated Cas9 (dCas9) and a guide RNA that specifically binds to a target gene; And a third step of treating the reactant with a fluorescent labeling factor.

The inventors of the present invention have completed a method of specifically detecting a target gene with high sensitivity without having to separate the gene from the cell lysate in detecting the target gene.

The method for detecting a target gene of the present invention includes a first step of lysing a biological sample containing the target gene.

The first step may provide a state in which a target gene present in the biological sample may bind to a complex consisting of inactivated Cas9 and guide RNA.

In the present invention, the complex consisting of the inactivated Cas9 (dCas9) and the guide RNA specifically binding to the target gene may be formed before performing the method of detecting the target gene, but is not limited thereto. In practice, dCas9 and guide RNA may be formed by reacting the sample sequentially or together.

In the present invention, the term "dissolution" is to put the target gene in a state capable of binding to the complex of the present invention, the lysis can be carried out according to a known method.

As used herein, the term "biological sample" refers to any sample containing a target gene. The biological sample can be any tissue or body fluid obtained from a subject comprising a target gene.

The biological sample may be a sputum, blood, serum, plasma, blood cells (eg, white blood cells), tissue, biopsy sample, smear sample, wash sample, swab sample, cell-containing body fluid, flow nucleic acid, urine, peritoneal fluid, and pleural effusion. , Cerebrospinal fluid, feces, lacrimal fluid or cells therefrom. The biological sample may also include tissue sections taken for histological purposes, ie frozen or fixed sections or microdissected cells or extracellular parts thereof. The biological sample can be obtained in a manner that does not harm the subject.

The method for detecting a target gene of the present invention comprises a second step of reacting the lysate with a complex consisting of inactivated Cas9 (dCas9) and guide RNA specifically binding to the target gene.

The second step is a step of reacting the complex with a lysate of a biological sample containing a target gene, and through the reaction includes a conjugate combined with the target gene, genes other than the unreacted target gene and complex Reactants can be provided.

In the present invention, the terms "inactivated Cas9" and "guide RNA" are the same as described above.

The method for detecting a target gene of the present invention includes a third step of separating the complex from the reactant.

The third step is the step of separating the complex from the reactants of the complex and the lysate containing the dCas9 protein, the dCas9 protein may include an affinity tag used for separation or purification according to the purpose.

The affinity tag is the same as described above.

The method for detecting a target gene of the present invention includes a fourth step of treating a fluorescent labeling factor in an isolated complex.

In the fourth step, a fluorescent labeling factor is processed on the separated complex, so that only the complex associated with the target gene among the complexes specifically has a fluorescence signal, thereby detecting the target gene.

The complex associated with the target gene has a double-stranded DNA, unlike the complex not bound to the target gene, and the complex associated with the target gene can be identified as a fluorescent marker that can specifically bind to the double-stranded DNA. .

In the present invention, the term "fluorescent labeling factor" is the same as described above.

In the present invention, the method for detecting the target gene may not undergo a separate purification process for the lysate of the biological sample of the first step.

In one embodiment of the present invention, after lysing the cells containing the target gene, the lysate was reacted with the complex of sgRNA and dCas9 without separating the target gene. Thereafter, the complex was separated by magnetic beads using His tag of dCas9 protein, and the eluted sample was treated with SYBR green I. As a result of observing the fluorescence signal, it was confirmed that a particularly strong fluorescence signal was detected in the target gene, and it was found that the target gene could be specifically detected through the above method (FIG. 5). Through this, it was found that the target gene can be specifically detected with high sensitivity without going through the step of separating the gene from the cell lysate.

The target gene detection method according to the present invention can detect a target gene with high sensitivity without performing PCR, and can be usefully used to quickly and accurately detect a target gene without a separate gene separation step.

1 is an electrophoresis result confirming that the target gene is cleaved upon the reaction of the complex of sgRNA / Cas9 and the target gene.

2 is an electrophoresis result confirming the change in the mobile phase of the target gene when the complex of sgRNA / dCas9 and the target gene in the reaction.

3 is a graph confirming the fluorescence signal using the complex of sgRNA / dCas9 and SYBR green I, a specific fluorescent signal appears in the gene of MRSA.

4 is a graph showing the intensity of the fluorescence signal according to the amount of the gene of MRSA of # 78 as a result of confirming the fluorescence signal using the complex of sgRNA / dCas9 and SYBR green I.

Figure 5 is a graph confirming that a specific fluorescent signal appears in the gene of MRSA without a separate gene purification process.

6 is a graph confirming the measured value of the LOD using the fluorescence signal of the lysate of MRSA.

7 is a diagram showing that the fluorescent signal is specifically expressed in the gene of MRSA through the micro array.

8 is a graph confirming that the fluorescent signal is specifically expressed in the gene of MRSA through the microarray.

Hereinafter, the configuration and effects of the present invention through the embodiments will be described in more detail. However, the following examples are merely to illustrate the invention is not limited to the contents of the present invention by the following examples.

Example  One: Cas9  or with dCas9 sgRNA  Target specificity of the complex

Example 1-1 Cas9-sgRNA Complex

100 ng of # 1539 (SEQ ID NO: 1) and # 1545 (SEQ ID NO: 2) were reacted overnight at 37 ° C with 100 ng of mecA gene, 238.5 ng of Cas9 protein, and 10X reaction buffer, respectively. Thereafter, electrophoresis was performed using 1.2% agarose gel to confirm that the mecA gene was cleaved. The sequence of the sgRNA gene used for the reaction is shown in Table 1 below.

TABLE 1

(Underlined is target gene specific sequence; adjacent bold means PAM sequence)

As a result, the mecA gene was maintained in the group (NC) not treated with the sgRNA of # 1539 or # 1545, while the mecA gene was cleaved in the group treated with the sgRNA (FIG. 1). This suggests that sgRNA of the sgRNA and Cas9 complex binds to the target gene, and Cas9 can cleave the target gene.

Example 1-2: sgRNA / dCas9 Complex

100 ng of # 1539 and # 1545 sgRNA were reacted with 100 ng of mecA gene, 1.5 ug of dCas9 protein, and 10X reaction buffer at 37 ° C. for 1 hour 30 minutes. Thereafter, electrophoresis was performed using 0.8% agarose gel to confirm the mobility shift of the mecA gene.

As a result, in the group (NC) not treated with the sgRNA of # 1539 or # 1545, there was no change in the mobility of the mecA gene, whereas the group treated with the sgRNA showed a change in the mobility of the mecA gene (FIG. 2). This suggests that sgRNA binds to the target gene and the sgRNA / dCas9 complex can maintain the binding state with the target gene.

Example 2: Specific Detection of Target Genes

The complex of sgRNA / dCas9 and SYBR green I were used to determine whether the target gene could be specifically detected.

Specifically, MRSA (# 78, # 81, # 82 and # 84; # means patient number) or MSSA (# 85, # 88, # 94 and ATCC25923; # is obtained from a skin lesion sample of Yonsei University Dermatology patient. The sgRNA (# 1539) and dCas9 proteins of SEQ ID NO: 1, which are complementary to the gene of MRSA, were reacted with the gene isolated from the patient number). Isolation of the MRSA or MSSA gene was performed using a DNA purification kit (Wizard® Genomic DNA purification Kin, Promega). Subsequently, only the dCas9 protein was separated by Ni-NTA magnetic beads using His tag of dCas9 protein, and the sample eluted was treated with SYBR green I. After reacting for 20 minutes at room temperature, the fluorescence signal and intensity were observed with a microplate machine.

As a result, almost no fluorescence signal was detected in the gene of MSSA, whereas a very strong fluorescence signal was detected in the gene of MRSA (FIG. 3). Through this, it was found that the target gene can be specifically detected by using the complex of sgRNA / dCas9 and SYBR green I.

Example 3: Fluorescence intensity according to concentration of target gene

DNA was isolated from MRSA at # 78 using a DNA purification kit (Wizard® Genomic DNA purification Kin, Promega). Various amounts of isolated DNA in the range of 0 to 1000 ng were reacted with the dCas9 / sgRNA complex. SYBR green I was treated on the sample eluted by separating the reactants with Ni-NTA magnetic beads. The intensity of fluorescence was observed at 494 nm.

As a result, even when a very low concentration of the gene was treated, a fluorescence signal was observed, and as the amount of the treated MRSA gene increased, it was confirmed that a high intensity fluorescence signal appeared (FIG. 4). Through this, it was found that only a small amount of the gene can detect the target gene.

Example 4: Confirmation of detection of target gene using lysate of MRSA

Unlike Example 3, it was confirmed whether the target gene can be detected without purifying the gene in the lysate of MRSA.

Specifically, MRSA of # 78, # 81, # 82 or # 84 or MSSA of # 85, # 88, # 94 and ATCC25923 were incubated to have an optical density of 1.0, and then lysozyme and resources After the addition of taffin (lysostaphin) for 1 hour at 37 ℃, a sample of cell lysates was prepared without additional purification. After reacting the sample with the dCas9 / sgRNA complex, the sample was eluted by separating the reactant with Ni-NTA magnetic beads and treated with SYBR green I. After reacting for 20 minutes at room temperature, the fluorescence signal and intensity were observed with a microplate machine.

As a result, it was confirmed that high fluorescence signal appeared only in the group reacted with the cell lysate sample of MRSA in the same manner as in Example 2 (FIG. 5). Through this, it can be seen that using the dCas9 / sgRNA complex can specifically detect the target gene without a separate gene purification process.

Example 5: Determination of LOD with Lysate of MRSA

MRSAs with various optical densities were added with lysozyme and lysostaphin and reacted at 37 ° C. for 1 hour, thereby preparing samples of cell lysates without further purification. After reacting the sample with the dCas9 / sgRNA complex, the sample was eluted by separating the reactant with Ni-NTA magnetic beads and treated with SYBR green I. Observing the fluorescence signal and converting the optical density to the corresponding cfu / mL to measure the LOD, it was confirmed that exhibits a limit of detection (LOD) of 10 cfu / mL (Fig. 6).

Example 6: Confirmation of Target Gene Detection by Microarray

The epoxide functional glass slide (epoxide functional glass slide) was reacted with 0.01M AB-NTA free fatty acid (in 0.1M Tris-HCL, pH 8.0) overnight at room temperature (RT), washed with ethanol and dried. Then, overnight reaction was performed at 0.1 M of nickel chloride (in 0.1 M Tris-HCL, pH 8.0) at room temperature (RT), followed by washing with ethanol and drying, followed by Ni-NTA functional glass slide (Ni- NTA functional glass slide) was prepared.

Genes purified from MRSA or MSSA were reacted with the dCas9 / sgRNA complex for 2 hours at 37 ° C to prepare a sample to form a dCas9 / sgRNA / gene complex. The prepared composite sample was spattered on the prepared slide, and reacted for 1 hour at room temperature (RT), followed by washing three times. The slides were treated with 1X SYBR green I and imaged with ChemiDoc, followed by fluorescence signal analysis.

As a result, it was confirmed that no fluorescence signal was detected at the position where the complex was reacted with the MSSA gene, whereas a high fluorescence signal was detected at the position where the complex was reacted with the MRSA gene (Figs. 7 and 8). . This suggests that the target gene can be specifically detected using the dCas9 / sgRNA complex.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto.

Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (9)

A first step of reacting a sample comprising a target gene with a complex consisting of inactivated Cas9 (dCas9) and a guide RNA that specifically binds to a target gene; A second step of separating the reactants; And A method for detecting a target gene, comprising the third step of processing the fluorescent marker. The method of claim 1, wherein the guide RNA is single chain guide RNA (sgRNA). The method of claim 1, wherein the inactivated Cas9 comprises an affinity tag. The tag of claim 3, wherein the affinity tag is a His tag, a Flag tag, an S tag, a Glutathione S-transferase (GST) tag, a Maltose binding protein (MBP) tag, a chitin binding protein (CMP) tag, an Avi tag, or a cal module. Calmodulin tags, polyglutamate tags, E tags, HA tags, myc tags, SBP tags, softtag 1, softag 3, strap tags, TC tags, A method for detecting a target gene, wherein the target gene is selected from the group comprising an Xpress tag, a biotin carboxyl carrier pretein (BCCP) tag, and a green fluorescent protein (GFP) tag. The method of claim 1, wherein the second step is performed using magnetic beads that bind to the tag. The method of claim 5, wherein the magnetic beads are Ni-NTA magnetic beads. The method of claim 1, wherein the fluorescent markers are SYBR green I, SYBR green II, SYBR gold, Oxazole yellow, YOYO, and Thiazole orange. Thiazole orange) is selected from the group comprising a method for detecting a target gene. Lysing the cell containing the target gene; A second step of reacting the lysate with a complex consisting of inactivated Cas9 (dCas9) and a guide RNA that specifically binds to a target gene; A third step of separating the reactants; And A method for detecting a target gene, comprising the fourth step of processing the fluorescent marker. The method of claim 8, wherein the method for detecting the target gene is characterized in that the lysate of the cell is not purified.

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