WO2023146370A1 - Composition and method for detecting nucleic acid based on fluorescence signal capable of specifically detecting egfr mutation - Google Patents

Abstract

The present invention relates to a CRISPR/Cas system capable of specifically amplifying EGFR mutant DNA, and a use thereof for diagnosing cancer. A CRISPR/Cas12a amplification system can be used to specifically and effectively amplify mutant DNA derived from a blood sample and thereby confirm mutations having a ratio of less than 0.5% that can be confused for sequencing errors.

Description

Fluorescent signal-based nucleic acid detection composition and method capable of specifically detecting EGFR mutation

It relates to a composition and method for detecting a nucleic acid based on a fluorescence signal capable of specifically detecting an EGFR mutation.

Patients with non-small lung cancer (NSCLC), known to have a higher mortality rate than other cancers, require early diagnosis for effective treatment. Given that a tyrosine kinase inhibitor, a targeted cancer treatment for non-small lung cancer (NSCLC), has been shown to have a strong therapeutic effect on EGFR mutation-positive NSCLC, L858R, a major biomarker for NSCLC, and early and stable detection of EGFR mutations of exon 19 deletion is very important for effective treatment.

Recently, the possibility of cancer diagnosis through the detection of DNA mutations in cell-free circulating DNA (cfDNA) released from tumor tissue has been demonstrated. Liquid biopsy-based diagnosis using blood samples is much less invasive and faster than tissue biopsy. There are simple advantages.

However, there are several obstacles to diagnosing cancer from cell-free circulating DNA (cfDNA).

First, a small amount of cfDNA in blood interferes with accurate diagnosis, requiring additional procedures such as PCR. Second, Next Generation Sequencing (NGS) still has an error rate of 0.5%, making it indistinguishable from rare mutant DNA. will be. In particular, the predominant concentration of normal DNA in blood samples is a major obstacle to sensitive detection of rare mutant DNA.

To address these issues, several studies have attempted to improve the sensitivity of oncogenic mutation diagnosis using CRISPR (Clustered regular interspaced short palindromic repeats) and CRISPR Associated Protein (Cas) systems.

Existing methods are based on the strategy of finding point mutations within the PAM motif sequence to disrupt PAM, but point mutations must be located in the PAM motif sequence, and some such as the EGFR L858R mutation without a PAM sequence (NGG) near the mutation. It has limitations that hinder its application to mutations.

Accordingly, it is necessary to develop a technology for a CRISPR system capable of detecting mutant DNA with high sensitivity by locating the mutant in a protospacer corresponding to the target DNA.

One aspect hybridizes to a target sequence and includes a guide polynucleotide comprising consecutive bases complementary to an epidermal growth factor receptor (EGFR) mutant gene sequence or a nucleic acid encoding the guide polynucleotide; A CRISPR/Cas effector protein, or a nucleic acid encoding the protein; and a single-stranded, EGFR mutation-detecting composition comprising a labeled detection nucleic acid that hybridizes with the guide polynucleotide.

Another aspect is to provide a kit for detecting an EGFR mutation comprising the composition.

Another aspect includes contacting a sample with the composition; And detecting a target sequence in the sample by measuring a detectable signal generated by cleavage of the single-stranded detection nucleic acid by the CRISPR / Cas effector protein. will be.

Another aspect is to provide a cancer diagnosis kit including the composition.

Another aspect is to provide a guide polynucleotide comprising a polynucleotide comprising any one of SEQ ID NOs: 2-6 or a polynucleotide having at least 95% homology to a polynucleotide of any one of SEQ ID NOs: 2-6.

Another aspect is to provide a method of diagnosing cancer or providing information related to cancer diagnosis, including contacting the composition with a sample.

One aspect hybridizes to a target sequence and includes a guide polynucleotide comprising consecutive bases complementary to an epidermal growth factor receptor (EGFR) mutant gene sequence or a nucleic acid encoding the guide polynucleotide; A CRISPR/Cas effector protein, or a nucleic acid encoding the protein; and a single-stranded, labeled detection nucleic acid that hybridizes with the guide polynucleotide.

Another aspect is to provide a kit for detecting an EGFR mutation comprising the composition.

In the present specification, the term "protospacer adjacent motif (PAM)" is a sequence recognized by the Cas protein of the CRISPR/Cas system. The protospacer sequence is a sequence located at the 5' or 3' end of the PAM sequence and directs binding of the PAM effector protein complex to the target locus of interest. Specifically, the PAM may include a 5' T-rich motif. The PAM may consist of a 2 to 10 bp DNA sequence, preferably a 2 to 6 bp DNA sequence.

As used herein, the term "guide polynucleotide" is a short synthetic RNA molecule used for genome editing based on the CRISPR system. A "guide polynucleotide" is a spacer sequence that binds to a target DNA. The gRNA molecule consists of a scaffold sequence for Cas binding, an endonuclease.

The guide nucleotide includes guide RNA, gRNA, or guide RNA. In addition, the guide polynucleotide includes crRNA. The crRNA is a partial sequence present within the crRNA that binds and/or interacts with tracrRNA and/or effector proteins. The crRNA may be a wild-type crRNA or an engineered crRNA. In this case, the crRNA may include a direct repeat sequence and a spacer, and the direct repeat sequence may be located at the 5' end of the spacer. In addition, the crRNA may be located at the 3' end of tracrRNA. The guide polynucleotide includes tracrRNA. The tracrRNA scaffold sequence is all or part of a tracrRNA sequence that binds and/or interacts with a crRNA and/or an effector protein.

The tracrRNA may be a wild-type tracrRNA or an engineered tracrRNA. The engineered crRNA or tracrRNA may be a sequence in which a part of the nucleotide sequence of the wild-type crRNA or tracrRNA is artificially modified (substituted, deleted, or inserted) or modified to be shorter than the wild-type crRNA or tracrRNA sequence. The guide polynucleotide may further include a linker. The linker is a sequence serving to connect the tracrRNA and crRNA. The linker may be a sequence of 1 to 30 nucleotides. In one embodiment, the linker may be 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, or 25 to 30 nucleotide sequences. For example, the linker may be a 5'-GAAA-3' sequence, but is not limited thereto. The guide polynucleotide may be an engineered guide RNA in which one or more nucleotide sequences are deleted, substituted, or added from the wild-type guide polynucleotide.

The guide sequence can be any polynucleotide sequence that has sufficient complementarity with the target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. The gRNA forms a complex with the Cas protein to bind to a protospacer adjacent motif (PAM) and a target polynucleotide sequence having a protospacer (sequence complementary to a portion of the gRNA). Guide RNA (gRNA) is the element that confers the target specificity of the gRNA:Cas protein complex.

In the present specification, the term "EGFR" can be used interchangeably with epidermal growth factor receptor as an abbreviation of Epidermal Growth Factor Receptor. EGFR is part of a group of tyrosine kinase receptors, also called HER or the erbB family, which includes EGFR (HER1/ErbB1), HER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4).

In one embodiment, the EGFR mutant gene sequence may be a guide polynucleotide linked to the 3' end of a protospacer adjacent motif (PAM). More specifically, the protospacer adjacent motif (PAM) may include a 5' T-rich motif.

As used herein, the term "mutation" refers to a case in which there is a permanent change in the nucleotide sequence of an existing gene. Depending on the effect on the structure of genetic material, mutations can occur when there is an abnormality in the structure or number of chromosomes (chromosomal abnormality) and when a change occurs on a small scale in the nucleotide sequence of a gene. Examples of abnormalities in the structure of chromosomes include deletion, duplication, inversion, and translocation. As used herein, the term "deletion mutation" refers to a case in which a portion of a chromosome is lost. As used herein, the term "point mutation" refers to a nucleotide sequence constituting a locus that occurs when one or more thousands of pairs are deleted.

The term "EGFR mutation" herein means G719S mutation, G179C mutation, G719A mutation, S720F mutation, T790M mutation, D761Y mutation, exon 19 deletion mutation, D770_N771 insertion mutation, V765A mutation, T783A mutation, S761I mutation, T790M mutation, V769L mutation , N771I mutation, L858R mutation, L861Q mutation, L861R mutation. Representative EGFR mutation L858R is a point mutation in which the 858th amino acid is changed from Leusine (L) to Arginine (Arginine, R), and is a mutation belonging to exon 21. Also, as a representative biomarker of non-small cell lung cancer, EGFR Exon 19 deletion mutant means that exon 19 of the gene encoding EGFR is deleted. Unlike the L858R point mutation, EGFR Exon19 deletion mutants appear in various deletion sizes, such as 9bp to 18bp of exon19.

In one embodiment, the cancer may have an EGFR mutation. The cancer may be a solid cancer. The solid cancer refers to an abnormal tissue mass originating from an organ. Solid cancers can be malignant. Different types of solid cancers are named according to the type of cells that form them. Types of solid cancer include sarcomas, carcinomas and lymphomas. Examples of solid cancers include adrenal cancer, anal cancer, anaplastic large cell lymphoma, angioimmunoblastic T cell lymphoma, B cell lymphoma, cholangiocarcinoma, bladder cancer, brain/CNS tumor, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, Tumors of the Ewing family, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (gist), gestational trophoblast disease, hepatosplenic T-cell lymphoma, Hodgkin's lymphoma, intravascular large B-cell lymphoma, renal cancer, laryngeal cancer and hypopharyngeal cancer, liver cancer, lung cancer (non-small cell and small cell), lung carcinoid lymphomatous granulomatosis, malignant mesothelioma, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, nodular marginal zone B-cell lymphoma, non-Hodgkin's lymphoma, oral and oropharyngeal cancer, osteosarcoma , ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, primary effusion lymphoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer (basal and squamous cell, melanoma and Merkel cell), small intestine cancer, gastric cancer, testicular cancer, thymic cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor. EGFR mutations can cause colorectal cancer, pancreatic cancer, glioma, head and neck cancer, and lung cancer, particularly non-small cell lung cancer (NSCLC), among others. According to one embodiment of the present invention, it is particularly effective for diagnosis of solid cancer selected from the group consisting of colorectal cancer, pancreatic cancer, glioma, head and neck cancer and lung cancer, and even more for diagnosis of non-small cell lung cancer (NSCLC).

In one embodiment, the guide polynucleotide may be a guide polynucleotide having a length of 20 bp to 50 bp consisting of a scafold sequence and a target sequence in order from the 5' end to the 3' end. In some embodiments, the guide sequence is about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 in length. , 29, 30, 35, 40, 45, 50, 75 bp or more. In some embodiments, the guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 or less nucleotides in length. The guide nucleotide sequence preferably has bases of 20 bp to 50 bp, more preferably 30 bp to 50 bp in length.

In one embodiment, the guide polynucleotide may further include a sequence that increases cleavage efficiency. In some embodiments, the sequence increasing the cleavage efficiency may be a T-rich tailing sequence. In some embodiments, the guide polynucleotide may include nucleotide sequences represented by SEQ ID NOs: 2 to 6. In the nucleotides represented by SEQ ID NOs: 2 to 6, T (Thymine) may be U (Uracil).

In one embodiment, the method comprises the step of measuring (e.g., a V-type CRISPR/Cas effector protein (e.g., a Cas12 protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e)-mediated ssDNA cleavage measuring a detectable signal that becomes). A V-type CRISPR/Cas effector protein (e.g., a Cas12 protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e), wherein the guide RNA is a V-type CRISPR/Cas effector protein (e.g., a Cas12 protein such as Cas12a, Cas12b , Cas12c, Cas12d, Cas12e), the detectable signal can be any signal generated when ssDNA is cleaved. For example, in some cases the measuring step may include gold nanoparticle based detection, fluorescence polarization, colloidal phase transition/dispersion, electrochemical detection, semiconductor-based sensing; For example, phosphatase can be used to generate a pH change after ssDNA cleavage reaction by releasing inorganic phosphatase into solution by opening 2'-3' cyclic phosphatase), and detection of labeled detector ssDNA. can include The readout of this detection method can be any convenient readout. Examples of possible readouts include a measurable amount of detectable fluorescence signal; Visual analysis of bands on the gel (e.g., bands representing cleaved product relative to uncleaved substrates), visual or sensor-based detection of the presence or absence of color (i.e., color detection methods), and electrical signal (or specific positive) presence or absence, but is not limited thereto.

The step of measuring can in some cases be quantitative in the sense that, for example, the amount of signal detected can be used to determine the amount of target DNA present in a sample. The step of measuring can in some cases be qualitative, for example in the sense that the presence or absence of a detectable signal can indicate the presence or absence of targeted DNA. In some cases, no detectable signal will be present (eg, above a given threshold level) unless the otherwise targeted DNA(s) are present above a certain threshold concentration. In some cases, the detection threshold can be titrated by varying the amount of CRISPR/Cas effector protein, guide RNA, sample volume and/or detector ssDNA (if one is used). Thus, for example, if it is desired to set up more than one response, as will be appreciated by those skilled in the art, multiple controls may be used, each set up to detect a different threshold level of target DNA, and thus such A series of reactions can be used to determine the amount of target DNA present in a sample.

In one embodiment, the methods of the present disclosure can be used to determine the amount of a target nucleic acid (eg, a sample comprising a target DNA and a plurality of non-target DNAs). Determining the amount of target DNA in the sample may include comparing the amount of detectable signal produced from the test sample to the amount of detectable signal produced from a reference sample. Determining the amount of target DNA in a sample includes measuring a detectable signal to generate a test measurement; measuring a detectable signal produced by the reference sample to produce a reference measurement; and comparing the test measurement to a reference measurement to determine the amount of target DNA present in the sample.

For example, in some cases, methods of the present disclosure for determining the amount of target DNA in a sample may include: a) a sample (eg, a sample comprising a target DNA and a plurality of non-target DNAs): i) a guide RNA that hybridizes with the target DNA, (ii) a CRISPR/Cas effector protein that cleave nucleic acids present in the sample, and (iii) a detection ssDNA; b) measuring a detectable signal generated by CRISPR/Cas effector protein-mediated ssDNA cleavage (eg, cleavage of the detector ssDNA) to generate a test measurement; c) measuring a detectable signal produced by the reference sample to produce a reference measurement; and d) comparing the test measurement to a reference measurement to determine the amount of target DNA present in the sample.

In some embodiments, the sensitivity of a subject composition and/or method can be increased by combining detection with nucleic acid amplification. In some cases, nucleic acids in the sample are amplified before, simultaneously with, or after contact with a CRISPR/Cas effector protein (eg, a Cas12 protein) that cuts the ssDNA. In some cases, nucleic acids in the sample are amplified concurrently with contact with a CRISPR/Cas effector protein (eg, Cas12 protein). Transfection of a CRISPR/Cas effector protein (eg, Cas12 protein) if all components are added simultaneously (amplification component and detection component, such as CRISPR/Cas effector protein, eg, Cas12 protein, guide RNA, and detector DNA). -cleavage activity is capable of starting to degrade the nucleic acid of the sample at the same time as the nucleic acid undergoes amplification.

A variety of amplification methods and components are known to those skilled in the art, and any convenient method may be used. Nucleic acid amplification includes polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), quantitative PCR (qPCR), reverse transcription qPCR (RT-qPCR), nested PCR, multiplex PCR, asymmetric PCR, touchdown PCR, random primer PCR, hemi-nested PCR, polymerase cycling assembly (PCA), colony PCR, ligase chain reaction (LCR), digital PCR, methylation specific-PCR (MSP), co-at low denaturation temperature Amplification-PCR (COLD-PCR), allele-specific PCR, intersequence-specific PCR (ISS-PCR), whole genome amplification (WGA), inverse PCR, and thermal asymmetric interlaced PCR (TAIL- PCR) may be included.

In some cases, amplification is isothermal amplification. The term “isothermal amplification” refers to a method of amplifying nucleic acids (eg, DNA) (eg, using an enzymatic chain reaction) that can use a single temperature incubation to eliminate the need for a thermal cycler. Isothermal amplification is a form of nucleic acid amplification that does not rely on thermal denaturation of target nucleic acids during the amplification reaction and thus may not require multiple rapid changes in temperature. Thus, isothermal nucleic acid amplification methods can be performed either within or outside of a laboratory environment. In combination with a reverse transcription step, these amplification methods can be used to amplify RNA isothermally.

Examples of isothermal amplification methods include loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), nucleic acid sequence- based amplification (NASBA), transcription-mediated amplification (TMA), nickase amplification reaction (NEAR), rolling circle amplification (RCA), multiple displacement amplification (MDA), ramification (RAM), circular helicase -Dependent Amplification (cHDA), Single Primer Isothermal Amplification (SPIA), Signal-Mediated Amplification of RNA Technology (SMART), Self-Sustained Sequence Replication (3SR), Genomic Exponential Amplification Reaction (GEAR) and Isothermal Multiple Displacement Amplification (IMDA) Including, but not limited to.

In some cases, the amplification is recombinase polymerase amplification (RPA). Recombinase polymerase amplification (RPA) uses two opposing primers (much like PCR) and uses three enzymes: recombinase, single-stranded DNA-binding protein (SSB) and strand-displacement polymerase. Use Recombinase pairs with oligonucleotide primers having homologous sequences in the double-stranded DNA, SSB binds to the displaced DNA strand to prevent displacement of the primer, and strand displacement polymerase initiates DNA synthesis, where the primer is bound to the target DNA.

In some cases, a subject method comprises a sample (eg, a sample comprising a target DNA and a plurality of non-target ssDNAs) comprising: i) a CRISPR/Cas effector protein; ii) guide RNA (or precursor guide RNA array); and iii) a detection DNA that is single-stranded and does not hybridize with the guide sequence of the guide RNA. For example, in some cases, a subject method comprises contacting a sample with a labeled single-stranded detection nucleic acid (detection ssDNA) comprising a fluorescence-emitting dye pair; A CRISPR/Cas effector protein (e.g., a Cas12 protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e) is activated (by binding to a guide RNA in the context of a guide RNA that hybridizes to a target DNA), followed by a labeled detector ssDNA. cut; A detectable signal to be measured is produced by the fluorescence-emitting dye pair. For example, in some cases, the subject methods include contacting the sample with labeled detection ssDNA comprising a fluorescence resonance energy transfer (FRET) pair or a quencher/fluor (quencher/fluor) pair or both. In some cases, a subject method comprises contacting a sample with a labeled detection ssDNA comprising a FRET pair. In some cases, the subject methods include contacting the sample with a labeled detection ssDNA comprising a fluorine/quencher pair.

Fluorescence-emitting dye pairs include FRET pairs or quencher/fluor pairs. In both the case of the FRET pair and the quencher/fluorine pair, the emission spectrum of one of the dyes overlaps the region of the absorption spectrum of the other dye in the pair. As used herein, the term "fluorescence-emitting dye pair" is a generic term used to include both a "fluorescence resonance energy transfer (FRET) pair" and a "quencher/fluorine pair", both of which are further hereinafter. discuss in detail The term “fluorescence-emitting dye pair” is used interchangeably with the phrase “FRET pair and/or quencher/fluorine pair”.

In some cases (e.g., when the detection ssDNA contains a FRET pair) the labeled detection ssDNA produces an amount of detectable signal before being cleaved, and the amount of detectable signal measured is the amount of the detected signal when the labeled detection ssDNA is cleaved. is reduced In some cases, the labeled detection ssDNA generates a first detectable signal before being cleaved (e.g., from a FRET pair) and the first detectable signal is generated when the labeled detection ssDNA is cleaved (e.g., from a quencher/fluor pair). 2 Generates a detectable signal. Thus, in some cases, the labeled detection ssDNA includes a FRET pair and a quencher/fluor pair.

A donor-acceptor pair (a FRET donor moiety and a FRET acceptor moiety) is referred to herein as a "FRET pair" or a "signal FRET pair". Thus, in some cases, when one signal partner is a FRET donor moiety and the other signal partner is a FRET acceptor moiety, the target labeled detection ssDNA contains two signal partners (signal pairs). Thus, a target labeled detector ssDNA containing such a FRET pair (a FRET donor moiety and a FRET acceptor moiety) produces a detectable signal (the FRET signal) when the signal counterpart is very proximal (e.g., while on the same RNA molecule). ), but when the counterpart is separated (e.g., after cleavage of an RNA molecule of a CRISPR/Cas effector protein (e.g., a Cas12 protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e)) the signal decreases ( or absent).

FRET donor and acceptor moieties (FRET pairs) will be known to those skilled in the art, and any convenient FRET pair (eg, any convenient donor and acceptor moiety pair) may be used.

In some cases, a detectable signal is generated when the labeled detector ssDNA is cleaved (eg, in some cases, the labeled detector ssDNA includes a quencher/fluor pair). One signal partner of a signal quenching pair produces a detectable signal, and the other signal partner is a quencher moiety that quenches the detectable signal of the first signal partner (i.e. when the signal partners are proximal to each other, e.g. The quencher moiety quenches the signal of the signal moiety such that the signal from the signal moiety is reduced (quenched) when the signal counterpart of the signal pair is proximal).

For example, in some cases, the amount of detectable signal is increased when the labeled detection ssDNA is cleaved. In some cases, e.g., when both signals are present on the same ssDNA molecule prior to cleavage by the CRISPR/Cas effector protein, e.g., the signal exhibited by one signal partner (signal moiety) is the signal exhibited by the other signal partner. (quencher signal moiety). Such signal pairs are referred to herein as "quencher/fluor pairs", "quenching pairs" or "signal quenching pairs". For example, in some cases, one signal counterpart (e.g., a first signal counterpart) is a signal moiety that produces a detectable signal that is quenched by a second signal counterpart (e.g., a quencher moiety) . Thus, the signal partners of this quencher/fluor pair will produce a detectable signal when the partners are separated (e.g., after cleavage of the detector ssDNA by the V-type CRISPR/Cas effector protein), but when the partners are very proximal ( eg, before cleavage of the detection ssDNA by the CRISPR/Cas effector protein) the signal will be quenched.

The quencher moiety can quench the signal to varying degrees from the signal moiety (eg, prior to cleavage of the detection ssDNA by the CRISPR/Cas effector protein). In some cases, a quencher moiety is a signal if the signal detected in the presence of the quencher moiety (when the signal counterparts are proximal to each other) is no more than 95% of the signal detected in the absence of the quencher moiety (when the signal counterparts are separated). Quench the signal from the moiety. For example, in some cases, the signal detected in the presence of the quencher moiety is 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less of the signal detected in the absence of the quencher moiety. or less, 30% or less, 20% or less, 15% or less, 10% or less, or 5% or less. In some cases, no signal (eg, above background) is detected in the presence of the quencher moiety.

In some cases, the signal detected in the absence of the quencher moiety (when the signal partners are separated) is at least 1.2-fold greater than the signal detected in the presence of the quencher moiety (when the signal partners are proximal to each other) (e.g., at least 1.3x, at least 1.5x, at least 1.7x, at least 2x, at least 2.5x, at least 3x, at least 3.5x, at least 4x, at least 5x, at least 7x, at least 10x, at least 20x, or at least 50 times higher).

In some cases, the signal moiety is a fluorescent label. In some such cases, the quencher moiety quenches the signal from the fluorescent label (light signal) (eg, by absorbing energy in the label's emission spectrum). Thus, when the quencher moiety is not in proximity to the signal moiety, emission (signal) from the fluorescent label is detectable because no signal is absorbed by the quencher moiety. Any convenient donor acceptor pair (signal moiety/quencher moiety pair) may be used, and many suitable pairs are known in the art.

In some cases, the quencher moiety absorbs energy from the signal moiety (also referred to herein as a "detectable label") and then emits a signal (eg, light at a different wavelength). Thus, in some cases, the quencher moiety is the signal moiety itself (e.g., the signal moiety can be 6-carboxyfluorescein, while the quencher moiety can be 6-carboxy-tetramethylrhodamine ), in some cases the pair may also be a FRET pair. In some cases, the quencher moiety is a dark quencher. The dark quencher absorbs the excitation energy and dissipates the energy in a different way (eg as heat). Thus, the dark quencher has minimal or no fluorescence of its own (it emits no fluorescence).

Examples of fluorescent labels include Alexa Fluor® dye, ATTO dye (e.g., ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G , ATTO 542, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rho11, ATTO Rho12, ATTO Thio12, ATTO Rho101, ATTO 590, ATTO 594, ATTO Rho13, ATTO 610, ATTO 620, ATTO Rho14, ATTO 633, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740), a DyLight dye, a cyanine dye (eg, Cy2, Cy3, Cy3.5, Cy3b, Cy5, Cy5. 5, Cy7, Cy7.5), FluoProbes dye, Sulfo Cy dye, Seta dye, IRIS dye, SeTau dye, SRfluor dye, Square dye, Fluorescein isothiocyanate ( FITC), tetramethylrhodamine (TRITC), Texas Red, Oregon Green, Pacific Blue, Pacific Green, Pacific Orange, Quantum Dots and Tethered (tethered) fluorescent proteins, but are not limited thereto.

In some cases, the detectable label is an Alexa Fluor® dye, an ATTO dye (e.g., ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO 532 , ATTO Rho6G, ATTO 542, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rho11, ATTO Rho12, ATTO Thio12, ATTO Rho101, ATTO 590, ATTO 594, ATTO Rho13, ATTO 610, ATTO 620, ATTO Rho14, ATTO 633, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740), a DyLight dye, a cyanine dye (e.g. Cy2, Cy3, Cy3.5, Cy3b, Cy5 , Cy5.5, Cy7, Cy7.5), fluoroprobe dye, sulfo Cy dye, theta dye, IRIS dye, SeTau dye, SRfluor dye, square dye, fluorescein (FITC), tetramethylrhodamine (TRITC), and a fluorescent label selected from Texas Red, Oregon Green, Pacific Blue, Pacific Green and Pacific Orange.

In some cases, the detectable label is an Alexa Fluor® dye, an ATTO dye (e.g., ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO 532 , ATTO Rho6G, ATTO 542, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rho11, ATTO Rho12, ATTO Thio12, ATTO Rho101, ATTO 590, ATTO 594, ATTO Rho13, ATTO 610, ATTO 620, ATTO Rho14, ATTO 633, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740), a DyLight dye, a cyanine dye (e.g. Cy2, Cy3, Cy3.5, Cy3b, Cy5 , Cy5.5, Cy7, Cy7.5), fluoroprobe dye, sulfo Cy dye, theta dye, IRIS dye, SeTau dye, SRfluor dye, square dye, fluorescein (FITC), tetramethylrhodamine (TRITC), and a fluorescent label selected from Texas Red, Oregon Green, Pacific Blue, Pacific Green, Pacific Orange, quantum dots and tethered fluorescent proteins.

Examples of ATTO dyes include, but are not limited to: ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 542, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rho11, ATTO Rho12, ATTO Thio12, ATTO Rho101, ATTO 590, ATTO 594, ATTO Rho13, ATTO 610, ATTO 620, ATTO Rho14, ATTO 633, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665, ATTO 680, ATTO 700, ATTO 725 and ATTO 740.

Examples of Alexa Fluor dyes include, but are not limited to: Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 500, Alexa Fluor® 514, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor ( Alexa Fluor (registered trademark) 610, Alexa Fluor (registered trademark) 633, Alexa Fluor (registered trademark) 635, Alexa Fluor (registered trademark) 647, Alexa Fluor (registered trademark) 660, Alexa Fluor (registered trademark) ) 680, Alexa Fluor (registered trademark) 700, Alexa Fluor (registered trademark) 750, Alexa Fluor (registered trademark) 790, etc.

Examples of quencher moieties include dark quencher, black hole quencher (registered trademark) (BHQ®) (e.g., BHQ-0, BHQ-1, BHQ-2, BHQ -3), Qxl Quencher, ATTO Quencher (eg ATTO 540Q, ATTO 580Q, and ATTO 612Q), Dimethylaminoazobenzenesulfonic Acid (Dabsyl), Iowa Black RQ, Iowa Black FQ, IRDye QC-1, QSY dyes (e.g., QSY 7, QSY 9, QSY 21), AbsoluteQuencher, Eclipse, and metal clusters such as gold nanoparticles, etc. don't

In some cases, the quencher moiety is a dark quencher, black hole quencher® (BHQ®) (e.g., BHQ-0, BHQ-1, BHQ-2, BHQ-3), Qxl quencher, ATTO quencher (e.g. ATTO 540Q, ATTO 580Q and ATTO 612Q), dimethylaminoazobenzenesulfonic acid (dansyl), Iowa Black RQ, Iowa Black FQ, IRDye QC-1, QSY dyes (e.g. QSY 7, QSY 9, QSY 21), Absolute Quencher, Eclipse and Metal Cluster.

Examples of ATTO quenchers include, but are not limited to, ATTO 540Q, ATTO 580Q, and ATTO 612Q. Examples of Black Hole Quencher® (BHQ®) include BHQ-0 (493 nm), BHQ-1 (534 nm), BHQ-2 (579 nm) and BHQ-3 (672 nm) , but not limited to these.

In some cases, cleavage of the labeled detector ssDNA can be detected by measuring the colorimetric readout. For example, liberation of a fluorophore (eg, liberation from a FRET pair, liberation from a quencher/fluor pair, etc.) can result in a wavelength shift (and thus color shift) of the detectable signal. Thus, in some cases, cleavage of the detector labeled ssDNA of interest can be detected by color-shift. This shift can be expressed as loss of the amount of one color signal (wavelength), gain of the amount of another color, change in the assignment of one color to another, and the like.

As used herein, the term "vector" is a DNA delivery vehicle essential for recombinant DNA technology used to obtain, propagate (amplify), or express a protein so that a gene can be conveniently used. The vector may be a viral vector or a non-viral vector. For example, the viral vector may be a retroviral (retrovirus) vector, a lentiviral (lentivirus) vector, an adenoviral (adenovirus vector), or an adeno-associated viral vector. (adeno-associated virus; AAV) vector), vaccinia viral (vaccinia virus) vector, poxviral (poxvirus) vector and herpes simplex viral (herpes simplex virus) vector ) may be one or more viral vectors selected from the group consisting of

The non-viral vector may be a plasmid, phage, naked DNA, DNA complex, mRNA (transcript) or PCR amplicon. For example, the plasmid consists of the pcDNA series, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19. It may be selected from the group. For example, the phage may be selected from the group consisting of λgt4λB, λ-Charon, λΔz1, and M13.

The vectors may optionally further contain regulatory/control elements, promoters and/or additional expression elements. The regulation/control element may be operably linked to a sequence encoding each element included in the vector (ie, a nucleic acid encoding a guide RNA and/or a nucleic acid encoding an effector protein). The regulatory/control elements include enhancers, introns, termination signals, polyadenylation signals, Kozak consensus sequences, Internal Ribosome Entry Sites (IRES), splice acceptors, 2A sequences, and/or It may be a replication origin (replication origin), but is not limited thereto. The origin of replication may be the f1 origin of replication, the SV40 origin of replication, the pMB1 origin of replication, the adeno origin of replication, the AAV origin of replication, and/or the BBV origin of replication. The vector may optionally contain a promoter. The promoter may be operably linked to a sequence encoding each component included in the vector (ie, a nucleic acid encoding a guide RNA and/or a nucleic acid encoding an effector protein). The promoter is not limited as long as it can appropriately express a sequence encoding each component included in the vector (ie, a nucleic acid encoding a guide RNA and/or a nucleic acid encoding an effector protein). In one embodiment, the promoter sequence may be a promoter that promotes transcription of RNA polymerase (eg, pol I, pol II, or pol III). For example, the promoters include the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter, adenovirus major late promoter (Ad MLP), herpes simplex virus (HSV) promoter, CMV immediate early promoter region (CMVIE), and the like. cytomegalovirus (CMV) promoter, chicken b-actin (CBA) promoter, rous sarcoma virus (RSV) promoter, human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497 - 500 (2002)), enhanced It may be one of the U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep 1;31(17)), the human H1 promoter (H1) and the 7SK promoter (7SK).

The vector may optionally contain additional expression elements. In addition to the nucleic acid encoding the guide RNA, the vector may include a nucleic acid sequence encoding additional expression elements that a person skilled in the art would like to express as needed. For example, the additional expression elements include herbicide resistance genes such as glyphosate, glufosinate ammonium, or phosphinothricin; or an antibiotic resistance gene such as ampicillin, kanamycin, G418, bleomycin, hygromycin or chloramphenicol.

In one aspect, the guide polynucleotide; and a Cas protein or a polynucleotide encoding the same, and provides a CRISPR/Cas complex capable of cleaving a nucleic acid containing an EGFR mutant gene sequence.

The term "CRISPR/Cas system" herein is derived from the bacterial adaptive immune system for bacteriophages, which are particularly used as powerful tools for DNA cleavage. All CRISPR-Cas systems have target specificity through CRISPR RNA (crRNA), which can be slightly modified into guide RNA (gRNA) when used experimentally. A spacer of crRNA recognizes and binds to a target sequence, and at this time, the target sequence must be adjacent to a sequence called Protospacer Adjacent Motif (PAM). After binding of the crRNA to the target, the Cas protein cuts the target DNA. Therefore, the Cas protein can be engineered to operate on a desired target simply by changing the sequence of the spacer portion of the crRNA or gRNA.

In one embodiment, the Cas protein may correspond to Class 2. Specifically, the Cas protein corresponding to Class 2 may correspond to type V. More specifically, the Cas protein corresponding to the type V may be an effector protein selected from the group consisting of Cas12a, mgCas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, and Cas12i.

Another aspect includes contacting a sample with the composition; And detecting a target sequence in the sample by measuring a detectable signal generated by cleavage of the single-stranded detection nucleic acid by the CRISPR / Cas effector protein. .

Another aspect provides a method of diagnosing cancer or providing information related to cancer diagnosis, comprising contacting the composition with a sample.

In one embodiment, the isolated patient sample may include cell free DNA (cfDNA).

As used herein, the term "cfDNA" is an abbreviation for circulating free DNA, and means a piece of DNA that is not present in the cell nucleus but is suspended in the blood. "Circulating free DNA" or "cell free DNA" and the like can be used interchangeably. Circulating free DNA (cfDNA) is a DNA fragment (50-200 bp) released into plasma. cfDNA can be used to describe the various forms of DNA that circulate freely in the bloodstream, including circulating tumor DNA (ctDNA), cell-free mitochondrial DNA (ccf mtDNA), and cell-free fetal DNA (cffDNA). Elevated levels of cfDNA are observed when cancer is advanced. Through cfDNA analysis, it can be used for cancer diagnosis and follow-up by confirming whether tumor-related mutations are included in the plasma DNA of cancer patients.

Another aspect provides a guide polynucleotide comprising a polynucleotide comprising any one of SEQ ID NOs: 2-6 or a polynucleotide having at least 95% homology to a polynucleotide of any one of SEQ ID NOs: 2-6.

Mutant DNA derived from a blood sample is specifically amplified through in-vitro digestion and PCR amplification using the CRISPR/Cas system using Cas nuclease according to one aspect, resulting in a 0.5 sequencing error that can be confused with sequencing errors. There is an effect that can identify mutations with a ratio of less than %.

1 is a schematic diagram explaining that exon19 deletion mutant DNA can be distinguished through crRNA matching normal DNA.

Figure 2 is a photograph of the results of in vitro cleavage analysis of normal DNA and Exon 19-deficient DNA using crRNA and LbCas12a matching normal DNA.

3 is a graph showing the results of performing CRISPR/Cas12a amplification three times on a DNA mixture in which Exon19 deletion mutant DNA was serially diluted to 1/100,000 of normal DNA.

4 is a graph showing the results of confirming Exon19 deletion mutations through CRISPR/Cas12a amplification among specimens in which Exon19 deletion was confirmed through tissue biopsy.

Figure 5 is a schematic diagram of a fluorescence signal-based nucleic acid detection method through the side effect of LbCas12a.

6 is a diagram explaining the mechanism by which crRNA for detecting exon19 deletion mutant DNA causes side effects.

7 is a graph showing results of detection of exon19 deletion mutant DNA synthesized through fluorescence signals induced from the LbCas12a/crRNA complex.

Figure 8 is a schematic diagram of Exon19 deletion patterns validated by clinical lung cancer patient samples.

9 shows two positive samples (P-1, P-11) confirmed to contain a subtype 1 deletion by NGS and two negative samples (N-25, N-25, This is the result of measuring the fluorescence signal of N-26).

10 shows two positive samples (P-9, P-10) confirmed to contain a subtype 2 deletion by NGS and two negative samples (N-25, N-25, This is the result of measuring the fluorescence signal of N-26).

11 shows two positive samples (P-4) identified as containing a subtype 3 deletion by NGS and two negative samples (N-25, N-26) identified as not containing the mutation. This is the result of measuring the fluorescence signal of

Figure 12 shows two positive samples (P-8) identified as containing a subtype 4 (subtype4) deletion by NGS and two negative samples (N-25, N-26) identified as not containing the mutation. This is the result of measuring the fluorescence signal of

Hereinafter, the present invention will be described in more detail with examples, but this is only illustrative and is not intended to limit the scope of the present invention. It is apparent to those skilled in the art that the embodiments described below may be modified within a range that does not deviate from the essential gist of the invention.

Reference Example 1. Purification and confirmation of LbCas12a recombinant protein

To express the LbCas12a recombinant protein, E. coli BL21(DE3) cells were transformed with the pET28a-LbCas12a (addgene No. #114070) bacterial expression vector. Cells were cultured in Luria Broth (LB) at 37 °C until the culture reached an O.D of 0.6 and the recombinant protein was 18 with the addition of isopropyl β-D-thiogalactoside (IPTG, GenDEPOT, Texas, USA) to a final concentration of 1 mM. Induction at °C for 18 hours. The medium was removed by centrifugation (4000 rpm, 30 min) of the cell culture, and the pelleted cells were lysed in lysis buffer (20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM β-mercaptoethanol (BioRad, California, U.S.A.). ), 1% Triton X-100 (Sigma) and 1 mM phenylmethylsulfonyl fluoride (Sigma, St. Louis, USA) were lysed by sonication (Qsonica, Newtown, USA).

The cell lysate obtained by the sonication was centrifuged at 20,000 x g for 10 minutes to remove cell debris, and the harvested soluble fraction was purified by mixing with Ni-NTA resin (Takara, Nojihigashi, Japan). The mixture was incubated in binding buffer (20 mM Tris-HCl, pH 8.0, 300 nM NaCl) for 1 hour at 4 °C with agitation. After incubation, the Ni-NTA resin was washed with 10 volumes of wash buffer. Next, the protein bound to the Ni-NTA resin was eluted with an elution buffer (20 mM Tris-HCl (pH 8.0), 300 nM NaCl, and 200 mM imidazole).

Eluted proteins were filtered through a Centricon filter (Amicon Ultra, Millipore, Burlington, USA). The purity of the recombinant protein was confirmed by SDS-PAGE (10%, Biorad, California, USA) and Coomassie blue staining (Biorad, California, USA).

Reference Example 2. In vitro transcription of crRNA

For in vitro transcription of crRNA, a DNA oligo containing the crRNA corresponding to the target DNA and the T7 promoter sequence was purchased from COSMO Genentech.

DNA oligo was prepared in T7 RNA polymerase (NEB, Massachusetts, U.S.A), 50 mM MgCl2, 100 mM NTPs (ATP, GTP, UTP, CTP), 10X RNA polymerase reaction buffer, Murine RNase inhibitor, 100 mM DTT and DEPC at 37°C for 8 hours. mixed while. To completely remove DNA oligos, the mixture was incubated with DNase at 37 °C for 1 hour, and RNA was purified using an RNA purification kit (RBC, New Taipei City, Taipei). Purity and concentration of purified RNA were measured using a Nanodrop™ 2000 Spectrophotometer (Thermo-Fisher, Massachusetts, USA). The purified RNA was aliquoted and stored at -80 °C.

Reference Example 3. Preparation of PCR amplicons and in vitro excision

A PCR amplicon with a partial sequence deletion in exon 19 was obtained from HEK293T genomic DNA by overlapping PCR using a DNA primer containing the mutation.

The purified recombinant Cas12a and crRNA designed to remove DNA other than mutant DNA were pre-mixed and incubated with the PCR amplicons for 1 hour at 37 °C. Then, the Cas12a/crRNA ribonucleoprotein complex was inactivated at 90 °C for 1 minute.

Cleavage of PCR amplicons was confirmed by agarose gel electrophoresis (2%).

Reference Example 4. Concentration of mutant DNA through CRISPR/Cas12a amplification

In order to specifically amplify mutant DNA, purified recombinant LbCas12a protein and artificially synthesized crRNA were pre-mixed and incubated with PCR amplicon for 1 hour at 37 °C did Then, the Cas12a/crRNA ribonucleoprotein complex was inactivated by incubation at 90 °C for 1 min.

After cleavage of normal DNA through the above process, the cleaved mixture was amplified by PCR amplification to uncleaved mutant DNA (denaturation at 98 ° C for 30 seconds). ), primer annealing at 58 °C for 30 sec, extension at 72 °C for 30 sec, 30 cycles).

For high throughput sequencing, nested PCR (denaturation at 98°C for 30 seconds, primer annealing at 58°C for 30 seconds, and extension at 72°C for 30 seconds) , 35 cycles) was used to link the enriched PCR production with the barcode sequence. High throughput sequencing was performed with an Illumina I-seq 100 sequencing machine (Illumina, California, USA).

The mutant DNA rate was calculated by analyzing the sequencing raw data through the CRISPR analyzer web tool.

Specifically, cell-free circulating DNA (cfDNA) was extracted from the plasma of lung cancer patients, and the extracted cfDNA was artificially synthesized with purified recombinant LbCas12a protein to specifically amplify mutant DNA. CrRNA was mixed to cleavage normal DNA (nolmal DNA). Uncleaved mutant DNA was amplified from the cleaved mixture by PCR amplification. High throughput sequencing was performed and sequencing raw data was analyzed through the CRISPR analyzer web tool.

Reference Example 5. CRISPR/Cas12a-based fluorescent reporter assay

Fluorescent signals using collateral effects of LbCas12a were confirmed through a fluorophore-quencher reporter assay. To form a stable complex, the purified LbCas12a recombinant protein (50 nM) and the crRNA corresponding to the mutated DNA sequence (50 nM) were pre-mixed in NEBuffer 2.1 and allowed to stand at room temperature for 5 minutes. After adding the DNA mixture and the FQ reporter (5`/6-FAM/TTATT/BHQ1/3`) to the LbCas12a/crRNA ribonuclease assembly mixture, a 96 well black plate was prepared. plate), and the fluorescence signal was measured for 1 hour (FAM) at 1 minute intervals. (FQ (λex: 483/30 nm, λem: 530/30 nm) Fluoroskan™ Microplate Fluorometer (Thermo-Fisher, Massachusetts, U.S.A).)

Example 1. crRNA design and activity confirmation for specifically amplifying EGFR exon19 deletion mutants

Example 1.1. Design of crRNA to specifically amplify EGFR exon19 deletion mutants

A crRNA was designed to specifically amplify the EGFR exon19 deletion mutant.

Specifically, unlike the L858R point mutation, exon19 deletion patterns appear in various deletion sizes, such as 9bp to 18bp. Accordingly, as shown in FIG. 1 , in the case of EGFR exon19 deletion mutants, normal DNA and exon19 deletion mutant DNA can be effectively distinguished without additionally introducing incorrect pairing. The composition of crRNA for specifically amplifying EGFR exon19 deletion mutants is shown in Table 1.

WT crRNA construct (SEQ ID NO: 1) AA TTTCT ACTAA GTGTA GAT GGAGA TGTTG CTTCT CTTAA TTT AA TTTCT ACTAA GTGTA GAT direct repeat sequence GGAGA TGTTG CTTCT CTTAA target sequence TTT Sequences that increase cleavage efficiency

Example 1.2. Confirmation of in vitro cleavage activity of crRNA for specific amplification of EGFR exon19 deletion mutant

A DNA fragment with a 15 bp deletion in the EGFR Exon19 locus was synthesized and an in vitro cleavage assay was performed with crRNA and LbCas12a that perfectly matched the normal DNA sequence. The results are shown in Figure 2.

As shown in Figure 2, the normal DNA-specific crRNA significantly cleaved the normal DNA, but the mutant DNA containing the 15bp deletion did not cut at all.

The above result means that, unlike L858R point mutated DNA, only normal DNA can be cut by distinguishing between normal DNA and Exon19 deletion mutant DNA without introducing mis-pairing into crRNA.

Example 2. CRISPR/Cas system designed to specifically amplify EGFR exon19 deletion mutant and confirmation of EGFR exon19 deletion mutant detection sensitivity

Example 2.1 CRISPR/Cas system designed to specifically amplify EGFR exon19 deletion mutants

In order to confirm that a very small amount of EGFR exon19 deletion mutant DNA can be specifically amplified, PCR is performed after the normal DNA is specifically digested in the same manner as in Reference Example 3 using the crRNA of Example 1 and LbCas12a. amplified.

Specifically, a 15 bp exon 19 deleted DNA fragment was diluted with a normal DNA fragment at a ratio of 1:100,000. CRISPR/Cas12a amplification, including in vitro digestion and PCR amplification, was performed on the diluted DNA mixture in triplicate each. Exon19 deletion mutation frequencies were calculated by high-throughput sequencing. The results are shown in FIG. 10 .

As shown in Figure 3, mutant DNA at a 1:100,000 dilution was not detected using conventional liquid biopsy. On the other hand, it was confirmed that the Exon19 deletion mutant DNA could be amplified up to 12.6% through 3 amplification through the CRISPR/Cas system according to an embodiment of the present invention.

The above result means that the measurement sensitivity can be secured through the CRISPR/Cas12a system according to one embodiment of the present invention for mutant DNA that cannot be measured using a general liquid biopsy (No amplification, N/A).

Example 2.2 Detection of Exon19 deletion mutation through CRISPR/Cas12a amplification in cfDNA of lung cancer patients

To confirm the effect of detecting Exon19 deletion mutant DNA in clinical samples, CRISPR/Cas12a amplification of Exon19 deletion mutant DNA was performed in cfDNA of lung cancer patients.

Specifically, cfDNA was extracted from the blood of 11 patients with Exon 19 deletion (+) and Exon 19 deletion (-) confirmed by tissue biopsy, and CRISPR/Cas12a amplification was performed. The results are shown in FIG. 4 .

As shown in FIG. 4, CRISPR/Cas12a amplification significantly reduced the deleted mutant DNA to 99.1% in six Exon 19 deletion (+) samples identified through tissue biopsies (P1, P4, P8, P9, P10 and P11). amplification was confirmed. Two of these samples (P4 and P9) showed less than 1% deletion rate before amplification, but were amplified to 91.4% and 93.4%, respectively, with CRISPR/Cas12a amplification.

The above result means that the CRISPR/Cas12a amplification system corresponding to one embodiment can effectively amplify mutant DNA derived from a blood sample and can be used as a useful tool for detecting EGFR mutant DNA.

Example 3. Confirmation of fluorescent signal amplification of EGFR exon19 deletion mutant DNA based on side effect of LbCas12a through CRISPR/Cas12a system

Example 3.1. Confirmation of fluorescence signal amplification of synthesized EGFR exon19 deletion mutant DNA

CRISPR/Cas12a amplification system according to one embodiment of the present invention to solve the problem of not being able to distinguish between a fluorescent signal and a background signal induced by a side effect of Cas12a protein when detecting a small amount of target DNA using a side effect of Cas12a protein The fluorescence signal amplification of the EGFR exon19 deletion mutant DNA synthesized using was confirmed.

Specifically, as shown in FIG. 5 , when LbCas12a/crRNA recognizes a target DNA, non-specific ssDNase, which is a side effect of LbCas12a, is activated. The activated LbCas12a/crRNA ribonucleoprotein non-specifically cuts the single-stranded DNA containing the FQ ssDNA reporter mixed according to Reference Example 5. The amount of target DNA was quantified by the fluorescence signal derived from the cleaved FQ reporter. As shown in FIG. 6 , the fluorescence signal of the EGFR exon19 deletion mutant DNA was detected using crRNA matching the exon19 deletion DNA sequence at 37 °C for 20 minutes. Tables 2 and 3 show the constructs of crRNAs corresponding to various types of exon19 deletion DNA sequences, and among them, the fluorescence signal detection results of EGFR exon19 deletion mutant DNA with the crRNA corresponding to SEQ ID NO: 2 are shown in FIG. . In FIG. 7, "Blank" indicates that only Cas12a/crRNA ribonucleic acid protein was cultured without DNA.

crRNA construction matching exon19 deletion DNA sequence (SEQ ID NO: 2) AA TTTCT ACTAA GTGTA GAT GGAGA TGTCT TGATA GCGAC TTT AA TTTCT ACTAA GTGTA GAT direct repeat sequence GGAGA TGTCT TGATA GCGAC target sequence TTT TTT sequence

As shown in FIG. 7, the exon19 deletion mutant DNA synthesized as a result of detecting a fluorescent signal using crRNA matching the exon19 deletion DNA sequence without an amplification procedure using the CRISPR/Cas12a system according to one embodiment of the present invention is converted into normal DNA. It was confirmed that the signal was indistinguishable from the background signal except for the DNA mixture diluted 1/10 in . On the other hand, when CRISPR/Cas12a amplification was used, a clear fluorescence signal was observed from the background signal in all mixtures, including mixtures in which the synthesized exon19 deletion mutant DNA was diluted 1/100,000 with normal DNA.

The above result means that the CRISPR/Cas12a system according to one embodiment can effectively increase a low fluorescence signal that is indistinguishable from a background signal.

Example 3.2. Confirmation of fluorescent signal amplification of Exon19 deletion subpattern mutant DNA in patient cfDNA

To confirm that it can be applied to detection of rare mutant DNA in cfDNA, CRISPR/Cas12a amplification was performed to detect exon19 deletion mutant DNA in cfDNA of patients confirmed to have the mutation through NGS. In order to detect fluorescence signals of exon19 deletion mutant DNA in lung cancer patient samples, crRNAs corresponding to each of the four exon19 deletion mutant subpatterns found in NSCLC (non-small cell lung cancer) patients were designed and shown in FIG. 3 . As shown in FIG. 8 , exon 19 deletion mutant subtypes 1 to 4 (subtypes 1 to 4) represent forms in which 18 bp, 15 bp, 16+2 bp, and 9 bp of exon 19 are deleted, respectively. Fluorescence signals were measured at 37 °C for 40 minutes using LbCas12a and crRNA corresponding DNA sequences containing deletion subtypes of deletion mutants, and the results are shown in FIGS. 9 to 12 . In Figures 9-12, P represents a positive sample previously confirmed by tissue biopsy, and N represents a negative sample previously confirmed by tissue biopsy.

Subtype_1 crRNA construction (SEQ ID NO: 3) AA TTTCT ACTAA GTGTA GAT GATTC CTTGA TAGCG ACGGG TTT AA TTTCT ACTAA GTGTA GAT direct repeat sequence GATTC CTTGA TAGCG ACGGG target sequence TTT Sequences that increase cleavage efficiency Subtype_2 crRNA construction (SEQ ID NO: 4) AA TTTCT ACTAA GTGTA GAT GGAGA TGTTT TGATA GCGAC TTT AA TTTCT ACTAA GTGTA GAT direct repeat sequence GGAGA TGTTT TGATA GCGAC target sequence TTT Sequences that increase cleavage efficiency Subtype_3 crRNA construction (SEQ ID NO: 5) AA TTTCT ACTAA GTGTA GAT GGAAT CTTGA TAGCG ACGGG TTT AA TTTCT ACTAA GTGTA GAT direct repeat sequence GGAAT CTTGA TAGCG ACGGG target sequence TTT Sequences that increase cleavage efficiency Subtype_4 crRNA construction (SEQ ID NO: 6) AA TTTCT ACTAA GTGTA GAT GGAGA TGTTG GTTCC TTGAT TTT AA TTTCT ACTAA GTGTA GAT direct repeat sequence GGAGA TGTTG GTTCC TTGAT target sequence TTT Sequences that increase cleavage efficiency

9 shows two positive samples (P-1, P-11) confirmed to contain a subtype 1 deletion by NGS and two negative samples (N-25, N-25, This is the result of measuring the fluorescence signal of N-26). As shown in FIG. 9, no fluorescence signal was detected in all samples prior to CRISPR/Cas12a amplification, which is an embodiment of the present invention. On the other hand, the fluorescence signals of the two positive samples (P1 and P11) amplified through CRISPR/Cas12a amplification (3 times) were significantly detected, but the fluorescence signals of the negative samples (N25 and N26) were not detected.

10 shows two positive samples (P-9, P-10) confirmed to contain a subtype 2 deletion by NGS and two negative samples (N-25, N-25, This is the result of measuring the fluorescence signal of N-26). Fluorescent signals of clinical samples with subtype2 deletion were able to discriminate positive samples (P10) from negative samples (N25, N26) without CRISPR/Cas12a amplification. This seems to be because the P10 sample contains a high percentage (50.4%) of the mutant DNA already deleted before amplification. Nevertheless, it was confirmed that the fluorescence signal of the sample with subtype2 deletion was more clearly distinguished from the negative sample by CRISPR/Cas12a amplification.

11 shows two positive samples (P-4) identified as containing a subtype 3 deletion by NGS and two negative samples (N-25, N-26) identified as not containing the mutation. 12 shows two positive samples (P-8) confirmed to contain a subtype 4 (subtype4) deletion by NGS and two negative samples confirmed to not contain a mutation. It is the result of measuring the fluorescence signal of (N-25, N-26). As shown in FIGS. 11 and 12, the fluorescence signals of P4 and P8 samples in which subtype 3 and 4 deletion type mutations were positively confirmed were indistinguishable from negative samples (N25 and N26) before amplification with the CRISPR/Cas12a system. On the other hand, it was confirmed that the fluorescence signal was enhanced through CRISPR/Cas12a amplification.

The above result means that the CRISPR/Cas12a amplification system of one embodiment can effectively amplify mutant DNA derived from a blood sample. Therefore, it can be used for diagnosis of various diseases, and in addition to diagnosis, the distinction between normal DNA and mutant DNA can be applied to various fields such as cancer treatment through genome editing specific to pathogenic alleles.

Claims (20)

A guide polynucleotide that hybridizes to a target sequence and includes consecutive bases complementary to an epidermal growth factor receptor (EGFR) mutant gene sequence or a nucleic acid encoding the guide polynucleotide; A CRISPR/Cas effector protein, or a nucleic acid encoding the protein; and A composition for detecting an EGFR mutation comprising a single-stranded, labeled detection nucleic acid that hybridizes with the guide polynucleotide. The composition according to claim 1, wherein the single-stranded detection nucleic acid DNA comprises a fluorescence-emitting dye pair. The composition of claim 2, wherein the fluorescence-emitting dye pair is a FRET pair or a quencher/fluor pair. The method of claim 1, wherein the EGFR mutations are G719S mutation, G179C mutation, G719A mutation, S720F mutation, T790M mutation, D761Y mutation, exon 19 deletion mutation, D770_N771 insertion mutation, V765A mutation, T783A mutation, S761I mutation, T790M mutation, V769L Mutation, N771I mutation, L858R mutation, L861Q mutation, any one of the L861R mutation, the composition. The composition according to claim 1, wherein the guide polynucleotide comprises, in order from the 5' end to the 3' end, a direct repeating sequence and a guide sequence capable of hybridizing to the target sequence. The composition according to claim 5, wherein the guide polynucleotide further comprises a sequence increasing cleavage efficiency at the 3' end. The composition of claim 1, wherein the CRISPR/Cas effector protein is any one effector protein selected from the group consisting of Cas12a, mgCas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, and Cas12i. The composition according to claim 1, wherein the guide polynucleotide or the nucleic acid encoding the guide polynucleotide comprises a nucleotide sequence represented by any one of SEQ ID NOs: 2 to 6. A kit for detecting an EGFR mutation comprising the composition of claim 1. 10. The kit according to claim 9, further comprising a nucleic acid amplification component. contacting a sample with the composition of claim 1; and A method for detecting an EGFR mutation comprising detecting a target sequence in the sample by measuring a detectable signal generated by cleavage of the single-stranded detection nucleic acid by the CRISPR/Cas effector protein. The method of claim 11, wherein the sample is cell free DNA (cfDNA). The method of claim 11 , comprising determining the amount of the target sequence present in the sample. 14. The method of claim 13, wherein the determining step comprises: measuring the detectable signal to generate a test measurement; measuring a detectable signal produced by the reference sample to produce a reference measurement; and comparing the test measurement to the reference measurement to determine the amount of the target sequence present in the sample. The method of claim 11, wherein the CRISPR / Cas effector protein is any one effector protein selected from the group consisting of Cas12a, mgCas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, and Cas12i, detecting EGFR mutations method. 12. The method of claim 11, wherein measuring the detectable signal comprises one or more of gold nanoparticle-based detection, fluorescence polarization, colloidal phase transition/dispersion, electrochemical detection, and semiconductor-based sensing, EGFR mutation. How to detect. The method of claim 11, wherein the detection nucleic acid comprises a fluorescence-emitting dye pair. 18. The method of claim 17, wherein the fluorescence-emitting dye pair generates an amount of detectable signal before cleavage of the detection nucleic acid, and wherein the amount of detectable signal is reduced after cleavage of the detection nucleic acid. method. A guide polynucleotide comprising a polynucleotide comprising any one of SEQ ID NOs: 2 to 6 or a polynucleotide having at least 95% homology to a polynucleotide of any one of SEQ ID NOs: 2 to 6. contacting a sample with the composition of claim 1; and A method for diagnosing cancer or information about cancer diagnosis, comprising detecting a target sequence in the sample by measuring a detectable signal generated by cleavage of the single-stranded detection nucleic acid by a CRISPR/Cas effector protein. How to provide.

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