KR20180093636A - Method for detecting a target gene using a dCas9/gRNA complex and fluorescence marker - Google Patents
Method for detecting a target gene using a dCas9/gRNA complex and fluorescence marker Download PDFInfo
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- C12Q2563/107—Nucleic acid detection characterized by the use of physical, structural and functional properties fluorescence
Abstract
A first step of reacting a complex comprising a inactivated Cas9 (dCas9) and a guide RNA specifically binding to a target gene with a sample containing the target gene; A second step of separating the reactants; And a third step of treating the fluorescent marker.
The target gene detection method according to the present invention can detect a target gene with high sensitivity without performing PCR and can be used to rapidly and accurately detect a target gene without going through a separate gene separation step.
Description
The present invention relates to a method for detecting a target gene using a dCas9 / gRNA complex and a fluorescent marker, and more particularly, to a method for detecting a target gene using inactivated Cas9 (dCas9) and a guide RNA specifically binding to a target gene, A first step of reacting an included sample; A second step of separating the complex from the reactant; And a third step of treating the fluorescent marker.
Up to now, methods for detecting genes have been reported based on PCR methods. (ICT / RT-PCR) using direct PCR and immunoassay for immunoassay to remove the inhibiting factors and to increase the sensitivity. Has been developed and used for gene detection.
In particular, it is necessary to diagnose pathogen infection quickly and accurately in order to prevent the early coping and disease progression and spread to diseases caused by pathogenic bacteria infection. If pathogens can be diagnosed during incubation before the symptoms appear after infection, infection can be effectively prevented and the damage can be prevented.
However, the methods developed so far have a disadvantage in that preparation of a sample to extract a target gene is difficult in order to detect a gene to be identified, and it takes much time to cultivate the cell. Furthermore, there is a problem that abuse of drugs is constantly carried out due to lack of rapid diagnosis method and lack of accuracy. Therefore, there is still a need for rapid and accurate gene detection.
Accordingly, the present inventors have made intensive efforts to develop a rapid and accurate gene detection method. As a result, unlike conventional gene diagnosis methods in which PCR must be accompanied or genes are isolated and analyzed, a separate gene separation step and a PCR step are performed It is possible to detect the target gene with high sensitivity, and the present invention has been completed.
One object of the present invention is to provide a method for detecting a target gene, comprising the steps of: (1) reacting a complex comprising a inactivated Cas9 (dCas9) and a guide RNA specifically binding to a target gene with a sample containing a target gene; A second step of separating the complex from the reactant; And a third step of treating the fluorescent marker.
It is another object of the present invention to provide a method for screening a target gene, comprising: a first step of dissolving a cell containing a target gene; A second step of reacting the lysate with a complex of inactivated Cas9 (dCas9) and a guide RNA specifically binding to a target gene; A third step of separating the complex from the reactant; And a fourth step of treating the fluorescent marker.
In one aspect of the present invention, the present invention provides a method for detecting a target gene, comprising the steps of: (1) reacting a complex comprising a inactivated cas9 (dCas9) and a guide RNA specifically binding to a target gene with a sample containing the target gene; A second step of separating the complex from the reactant; And a third step of treating the fluorescent marker.
The inventors of the present invention have found that when a target gene is detected, a complex consisting of inactivated Cas9 (dCas9) and a guide RNA specifically binding to a target gene and a complex It is possible to overcome the disadvantages of low sensitivity immunoassay while simplifying the complicated procedure of molecular diagnostic method. Thus, a rapid and sensitive target gene specific detection method is completed.
The method for detecting a target gene of the present invention includes a first step of reacting a complex comprising a inactivated Cas9 (dCas9) and a guide RNA specifically binding to a target gene with a sample containing a target gene.
The first step is a step of reacting the complex with a sample containing two or more genes including a target gene, and the combination of the target gene and the complex bound to the complex, the gene other than the untreated target gene, Lt; RTI ID = 0.0 > non-conjugated < / RTI >
In the present invention, the complex consisting of the inactivated Cas9 (dCas9) and the guide RNA specifically binding to the target gene can be formed before performing the detection method of the target gene, but the present invention is not limited thereto, In doing so, dCas9 and guide RNA can be formed sequentially or together by reaction with the sample.
The term "sample" in the present invention means any sample containing a target gene.
In the present invention, the term "guide RNA" refers to RNA containing a sequence specifically binding to a target gene. The guide RNA of the present invention may form a complex with Cas9 protein. The guide RNA can be composed of crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA).
The crRNA can bind to the target gene.
The tracrRNA can bind to crRNA and change the structure of dCas9 protein.
Specifically, in the present invention, the guide RNA may be a single stranded sgRNA while maintaining the role of a crRNA and a tracrRNA.
The term "specific binding" in the present invention can be used in combination with hybridization.
The specific binding of the guide RNA to the target gene may mean that the guide RNA of the sequence complementary to the target gene hybridizes with the target sequence of the single strand of the target gene to form double stranded molecules (hybrid).
The sequence complementary to the target gene of the guide RNA can be hybridized with a part of the target gene, and the complementary sequence is 90% or more, specifically 95% or more, more specifically 100% complementary to a part of the target gene Sequence.
The term "inactivated Cas9" in the present invention is a Cas9 nuclease protein in which the function of nuclease is inactivated, and can also be named as dCas9. The production of the inactivated Cas9 protein can be produced by a conventional method of inactivating the activity of the nuclease, but is not limited thereto.
The Cas9 protein and its gene information can be obtained from a known database such as GenBank of National Center for Biotechnology Information (NCMBI).
The method of detecting a target gene of the present invention includes a second step of separating the complex in the reactant.
The second step is a step of separating the complex from the complex containing dCas9 protein and the reaction product of the sample. Therefore, the dCas9 protein may include an affinity tag used for separation or purification depending on the purpose.
The affinity tag includes a tag such as His tag, Flag tag, S tag, Glutathione S-transferase tag, Maltose binding protein (MBP) tag, Chitin binding protein (CMP) tag, Avi tag, A polygutamate tag, an E tag, an HA tag, a myc tag, an SBP tag, a
In the second step, the composite may be separated using a magnetic bead coupled to the tag, and the magnetic bead may be a Ni-NTA magnetic bead, but is not limited thereto.
The method of detecting a target gene of the present invention includes a third step of treating a fluorescent complex with a separated complex.
The third step is a step of treating the separated complex with a fluorescent marker. Only the complex bound to the target gene among the complexes can specifically detect a target gene by having a fluorescent signal.
The complex bound to the target gene can be identified as a complex with the target gene as a fluorescent marker capable of specifically binding to the double stranded DNA in a structure having double stranded DNA unlike the complex not bound to the target gene .
The term "fluorescent marker" in the present invention is a fluorescent substance used for coloring nucleic acid, and has a property of specifically binding to double-stranded DNA. In the present invention, the fluorescent marker may be specifically bound to the double-stranded DNA of the target gene bound to the complex.
The fluorescent marker may be a fluorescent marker 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, But is not limited thereto.
In one embodiment of the present invention, a complex of sgRNA and dCas9 was reacted with a sample containing various genes including a target gene, and then a magnetic bead conjugate was separated using a His tag of dCas9 protein. SYBR green I. As a result of observing the fluorescence signal, it was confirmed that a specifically strong fluorescence signal was detected in the target gene, and it was found that the target gene could be specifically detected by the above method (FIG. 2).
According to another aspect of the present invention, there is provided a method for detecting a target gene, comprising: a first step of dissolving a biological sample containing a target gene; A second step of reacting the lysate with a complex of inactivated Cas9 (dCas9) and a guide RNA specifically binding to a target gene; And a third step of treating the reactant with a fluorescent marker. The present invention also provides a method for detecting a target gene.
The present inventors completed a method for specifically detecting a target gene with high sensitivity without performing a step of isolating a gene from a cell lysate in detecting a target gene.
A method for detecting a target gene of the present invention includes a first step of dissolving a biological sample containing a target gene.
The first step may provide a condition in which a target gene present in the biological sample can bind to a complex consisting of Cas9 inactivated 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 can be formed before performing the detection method of the target gene, but the present invention is not limited thereto, In doing so, dCas9 and guide RNA can be formed sequentially or together by reaction with the sample.
The term "lysis" in the present invention means that the target gene is placed in a state capable of binding with the complex of the present invention, and the dissolution can be carried out according to a known method.
The term "biological sample" in the present invention means any sample containing a target gene. The biological sample may be any tissue or body fluid obtained from a subject containing the target gene.
The biological sample can be a biological sample such as sputum, blood, serum, plasma, blood cells (e.g., leukocytes), tissue, biopsy sample, smear sample, wash sample, swab sample, cell containing body fluid, fluid nucleic acid, urine, , Brain spinal fluid, feces, tears, or cells therefrom. The biological sample may also comprise a tissue section taken under a histological purpose, i. E. Frozen or fixed section or a microdissection cell or extracellular part thereof. The biological sample may be obtained in a manner that does not cause harm to the subject.
The method of detecting a target gene of the present invention comprises a second step of reacting the lysate with a complex of inactivated Cas9 (dCas9) and a guide RNA specifically binding to a target gene.
The second step is a step of reacting the complex with the lysate of the biological sample containing the target gene, and includes a combination of the target gene and the complex, a gene and a complex other than the unreacted target gene Lt; / RTI >
The term "inactivated Cas9" and "guide RNA" in the present invention are the same as described above.
The method of detecting a target gene of the present invention includes a third step of isolating the complex in the reactant.
The third step is a step of separating the complex from the reaction product of the complex containing dCas9 protein and the lysate. Therefore, the dCas9 protein may include an affinity tag used for separation or purification depending on the purpose.
The affinity tag is the same as described above.
The method of detecting a target gene of the present invention includes a fourth step of treating the separated complex with a fluorescent marker.
In the fourth step, a target gene can be detected by treating the separated complex with a fluorescent marker so that only the complex bound to the target gene has specific fluorescence signal among the complexes.
The complex bound to the target gene can be identified as a complex with the target gene as a fluorescent marker capable of specifically binding to the double stranded DNA in a structure having double stranded DNA unlike the complex not bound to the target gene .
The term "fluorescent marker" in the present invention is the same as described above.
In the present invention, the method of detecting the target gene may not require a separate purification process for the lysate of the biological sample of the first step.
In one embodiment of the present invention, after lysing cells containing the target gene, the lysate was reacted with a complex of sgRNA and dCas9, without isolating the target gene. After that, the complex was separated from magnetic beads using the 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 specifically strong fluorescence signal was detected in the target gene, and it was found that the target gene could be specifically detected by the above method (FIG. 4). This indicates that the target gene can be specifically detected with high sensitivity without 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 used to rapidly and accurately detect a target gene without going through a separate gene separation step.
FIG. 1A shows the result of electrophoresis in which a target gene is cleaved upon reaction of a sgRNA / Cas9 complex with a target gene.
Fig. 1B is a result of electrophoresis showing the change in the mobility of the target gene when the sgRNA / dCas9 complex is reacted with the target gene.
FIG. 2 is a graph showing fluorescence signals specifically observed in the MRSA gene as a result of confirming fluorescence signals using the sgRNA / dCas9 complex and SYBR green I.
FIG. 3 is a graph showing the intensity of fluorescence signals according to the amount of gene of
FIG. 4 is a graph showing that a fluorescent signal is specifically detected in the gene of MRSA without a separate gene purification process.
FIG. 5 is a graph showing measured values of LOD using fluorescence signals of a melt of MRSA.
FIG. 6A is a diagram showing that a fluorescent signal is specifically observed in a gene of MRSA through a microarray.
FIG. 6B is a graph showing that a fluorescence signal is specifically observed in the gene of MRSA through a microarray.
Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. The following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
Example One: Cas9 or dCas9 and sgRNA The target specificity of the complex
Example 1-1: Cas9 - sgRNA Complex
100 ng of sgRNA of # 1539 (SEQ ID NO: 1) and # 1545 (SEQ ID NO: 2) were each reacted with 100 ng of mecA gene, 238.5 ng of Cas9 protein and 10X reaction buffer overnight at 37 ° C. Thereafter, the mecA gene was cleaved by electrophoresis using 1.2% agarose gel. The sequence of the sgRNA gene used in the reaction is shown in Table 1 below.
[Table 1]
As a result, it was confirmed that the mecA gene was retained in the group (NC) not treated with sgRNA of # 1539 or # 1545, whereas the mecA gene was cleaved in the group treated with sgRNA (FIG. This suggests that sgRNA and Cas9 complex sgRNA bind to the target gene and Cas9 can cleave the target gene.
Example 1-2: sgRNA / dCas9 Complex
100 ng of sgRNA of # 1539 and # 1545 were reacted with 100 ng of mecA gene, 1.5 ug of dCas9 protein and 10X reaction buffer, respectively, at 37 캜 for 1.5 hours. Thereafter, electrophoresis was performed using 0.8% agarose gel to confirm whether mecA shifts the mecA gene.
As a result, it was confirmed that the mecA gene was not changed in the group (NC) treated with sgRNA of # 1539 or # 1545, while the mecA gene was found to be in the mobile phase in the group treated with sgRNA 1b). This suggests that the sgRNA / dCas9 complex can bind to the target gene by binding to the target gene.
Example 2: Specific detection of target gene
sgRNA / dCas9 complex and SYBR green I were used to confirm the ability to specifically detect the target gene.
Specifically, MRSA (# 78, # 81, # 82, and # 84; # are patient numbers) or MSSA (# 85, # 88, # 94, and
As a result, it was confirmed that a fluorescent signal was hardly detected in MSSA gene, whereas a very strong fluorescence signal was detected in MRSA gene (FIG. 2). Thus, it was found that the target gene could be specifically detected by using the complex of sgRNA / dCas9 and SYBR green I.
Example 3: fluorescence intensity according to the concentration of the target gene
DNA was isolated from # 78 MRSA using a DNA purification kit (Wizard® Genomic DNA purification Kin, Promega). Various amounts of isolated DNA ranging from 0 to 1000 ng were reacted with the dCas9 / sgRNA complex. The samples were eluted with Ni-NTA magnetic beads and treated with SYBR green I. Fluorescence intensity was observed at 494 nm.
As a result, fluorescence signals were observed even when the gene was treated at a very low concentration, and fluorescence signals of high intensity were observed as the amount of the treated MRSA gene was increased (FIG. 3). This indicates that the target gene can be detected with only a small amount of genes.
Example 4: MRSA's Melt Identification of the target gene used
Unlike in Example 3, the detection of the target gene was confirmed without purification of the gene in the MRSA lysate.
Specifically, the MRSA of # 78, # 81, # 82 or # 84 or the MSSA of # 85, # 88, # 94 and ATCC25923 were cultured so that the optical density was 1.0 and then lysozyme and resource After lysostaphin was added and incubated at 37 ° C for 1 hour, a sample of cell lysate was prepared without further purification. The sample was reacted with dCas9 / sgRNA conjugate, and the reaction was separated and eluted with Ni-NTA magnetic beads. The sample was treated with SYBR green I. After incubation at room temperature for 20 minutes, fluorescence signals and intensities were observed with a microplate machine.
As a result, it was confirmed that a fluorescence signal exhibited only in the group reacted with the cell lysate sample of MRSA as in the result of Example 2 (FIG. 4). This suggests that the use of the dCas9 / sgRNA complex can specifically detect the target gene without performing a separate gene purification step.
Example 5: MRSA's Melt Used Of LOD Measure
MRSA with various optical densities was reacted with lysozyme and lysostaphin at 37 ° C for 1 hour, and a sample of cell lysate was prepared without further purification. The sample was reacted with dCas9 / sgRNA conjugate, and the reaction was separated and eluted with Ni-NTA magnetic beads. The sample was treated with SYBR green I. The fluorescence signal was observed and LOD was measured in terms of optical density corresponding to cfu / mL. As a result, it was confirmed that LOD (limit of detection) was 10 cfu / mL (FIG. 5).
Example 6: Microarray Confirmation of target gene detection through
An epoxide functional glass slide was reacted with 0.01 M AB-NTA free fatty acid (0.1 M Tris-HCl, pH 8.0) at room temperature (RT) overnight, washed with ethanol and dried. Then, the reaction was carried out overnight with 0.1 M nickel chloride (in 0.1 M Tris-HCl, pH 8.0) at room temperature (RT), washed with ethanol and dried to obtain a Ni- NTA functional glass slide) was prepared.
The gene purified from MRSA or MSSA was reacted with dCas9 / sgRNA complex at 37 ° C for 2 hours to prepare a dCas9 / sgRNA / gene complex. The prepared composite sample was spun onto the prepared slide, allowed to react at room temperature (RT) for 1 hour, and then washed three times. Slides were treated with 1X SYBR green I, imaged with ChemiDoc, and analyzed for fluorescence signals.
As a result, it was confirmed that no fluorescent signal was detected at the spot where the complex was reacted with the MSSA gene, whereas a high fluorescence signal was detected at the spot where the complex was reacted with the MRSA gene (FIGS. 6A and 6B) . This indicates that the target gene can be specifically detected using the dCas9 / sgRNA complex.
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention.
It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.
<110> KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY
<120> Method for detecting a target gene using a dCas9 / gRNA complex and
가변 분자 마 marker
<130> P16-137-KRI
<160> 2
<170> KoPatentin 3.0
<210> 1
<211> 70
<212> DNA
<213> Artificial Sequence
<220>
<223>
Claims (9)
A second step of separating the reactants; And
And a third step of treating the fluorescent marker.
A second step of reacting the lysate with a complex of inactivated Cas9 (dCas9) and a guide RNA specifically binding to a target gene;
A third step of separating the reactants; And
And a fourth step of treating the fluorescent marker.
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| PCT/KR2017/001618 WO2018151339A1 (en) | 2017-02-14 | 2017-02-14 | Method for detecting target gene, using dcas9/grna complex and fluorescence marker |
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| KR102370880B1 (en) * | 2020-09-28 | 2022-03-04 | 한국생명공학연구원 | CRISPR/dCas9-mediated Surface-enhanced Raman Scattering Assay for Detecting Pathogen |
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| EP3650553B1 (en) * | 2018-11-07 | 2023-07-12 | Siemens Healthcare GmbH | Method for detection of specific nucleic acids |
| CN112063604A (en) * | 2020-09-15 | 2020-12-11 | 济南国科医工科技发展有限公司 | Functionalized dCas9 modified protein and application thereof in nucleic acid detection |
| KR102583531B1 (en) * | 2021-09-13 | 2023-10-06 | 한국과학기술연구원 | Composition for distinguishing of erythropoietin gene doping and use thereof |
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| KR100832738B1 (en) * | 2007-01-23 | 2008-05-27 | 포천중문의과대학교 산학협력단 | Methods for detecting a target gene using a peptide nucleic acid probe immobilized microarray |
| US9873907B2 (en) * | 2013-05-29 | 2018-01-23 | Agilent Technologies, Inc. | Method for fragmenting genomic DNA using CAS9 |
| US9322037B2 (en) * | 2013-09-06 | 2016-04-26 | President And Fellows Of Harvard College | Cas9-FokI fusion proteins and uses thereof |
| BR112017008931B1 (en) * | 2014-10-30 | 2022-02-01 | Ewha University - Industry Collaboration Foundation | Microfluidic device for target gene detection, method of manufacturing the same and method of detection using the same |
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| KR102370880B1 (en) * | 2020-09-28 | 2022-03-04 | 한국생명공학연구원 | CRISPR/dCas9-mediated Surface-enhanced Raman Scattering Assay for Detecting Pathogen |
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