WO2023146370A1 - Composition et procédé de détection d'acides nucléiques fondés sur un signal de fluorescence pouvant détecter spécifiquement une mutation egfr - Google Patents

Composition et procédé de détection d'acides nucléiques fondés sur un signal de fluorescence pouvant détecter spécifiquement une mutation egfr Download PDF

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WO2023146370A1
WO2023146370A1 PCT/KR2023/001357 KR2023001357W WO2023146370A1 WO 2023146370 A1 WO2023146370 A1 WO 2023146370A1 KR 2023001357 W KR2023001357 W KR 2023001357W WO 2023146370 A1 WO2023146370 A1 WO 2023146370A1
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mutation
atto
nucleic acid
dna
crispr
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Korean (ko)
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배태근
이재준
이효민
강승훈
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주식회사 페로카
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • It relates to a composition and method for detecting a nucleic acid based on a fluorescence signal capable of specifically detecting an EGFR mutation.
  • NSCLC non-small lung cancer
  • 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.
  • NGS Next Generation Sequencing
  • CRISPR Clustered regular interspaced short palindromic repeats
  • Cas CRISPR Associated Protein
  • 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.
  • EGFR epidermal growth factor receptor
  • 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.
  • 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.
  • EGFR epidermal growth factor receptor
  • Another aspect is to provide a kit for detecting an EGFR mutation comprising the composition.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 is the element that confers the target specificity of the gRNA:Cas protein complex.
  • 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).
  • 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.
  • PAM protospacer adjacent motif
  • 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.
  • 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.
  • 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.
  • 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.
  • 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, no
  • 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.
  • NSCLC non-small cell lung cancer
  • 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).
  • 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.
  • 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.
  • 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.
  • the guide polynucleotide may further include a sequence that increases cleavage efficiency.
  • the sequence increasing the cleavage efficiency may be a T-rich tailing sequence.
  • 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).
  • 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
  • a V-type CRISPR/Cas effector protein e.g., a Cas12 protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e
  • the guide RNA is a V-type CRISPR/Cas effector protein
  • the detectable signal can be any signal generated when ssDNA is cleaved.
  • the measuring step may include gold nanoparticle based detection, fluorescence polarization, colloidal phase transition/dispersion, electrochemical detection, semiconductor-based sensing;
  • 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.
  • 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.
  • 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).
  • 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.
  • 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
  • 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.
  • a sample eg, a sample comprising a target DNA and a plurality of non-target DNAs
  • a guide RNA that hybridizes
  • the sensitivity of a subject composition and/or method can be increased by combining detection with nucleic acid amplification.
  • 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.
  • nucleic acids in the sample are amplified concurrently with contact with a CRISPR/Cas effector protein (eg, Cas12 protein).
  • CRISPR/Cas effector protein eg, Cas12 protein
  • 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.
  • 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.
  • PCR polymerase chain reaction
  • RT-PCR reverse transcription PCR
  • qPCR quantitative PCR
  • RT-qPCR reverse transcription qPCR
  • nested PCR multiplex PCR
  • asymmetric PCR touchdown PCR
  • 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.
  • 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.
  • 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.
  • LAMP loop-mediated isothermal amplification
  • HDA helicase-dependent amplification
  • RPA recombinase polymerase
  • the amplification is recombinase polymerase amplification (RPA).
  • RPA recombinase polymerase amplification
  • SSB single-stranded DNA-binding protein
  • SSB strand-displacement polymerase
  • 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.
  • 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
  • a detectable signal to be measured is produced by the fluorescence-emitting dye pair.
  • 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.
  • a subject method comprises contacting a sample with a labeled detection ssDNA comprising a FRET pair.
  • 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.
  • 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
  • FRET fluorescence resonance energy transfer
  • quencher/fluorine pair both of which are further hereinafter. discuss in detail
  • fluorescence-emitting dye pair is used interchangeably with the phrase “FRET pair and/or quencher/fluorine 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
  • 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.
  • 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".
  • FRET pair a FRET donor moiety and a FRET acceptor moiety
  • signal FRET pair a signal partner
  • the target labeled detection ssDNA contains two signal partners (signal pairs).
  • 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).
  • RNA molecule of a CRISPR/Cas effector protein e.g., a Cas12 protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e
  • a Cas12 protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e
  • FRET donor and acceptor moieties 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.
  • 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
  • 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).
  • the amount of detectable signal is increased when the labeled detection ssDNA is cleaved.
  • the signal exhibited by one signal partner 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”.
  • one signal counterpart e.g., a first signal counterpart
  • a second signal counterpart e.g., a quencher moiety
  • 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).
  • 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.
  • 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.
  • the signal detected in the absence of the quencher moiety 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).
  • the signal moiety is a fluorescent label.
  • the quencher moiety quenches the signal from the fluorescent label (light signal) (eg, by absorbing energy in the label's emission spectrum).
  • 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.
  • 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).
  • 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.
  • 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).
  • 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.
  • ATTO dye e.g., ATTO 390, ATTO 425, ATTO 465, AT
  • 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.
  • 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., ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520, AT
  • fluorescein FITC
  • TRITC tetramethylrhodamine
  • 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., ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520, AT
  • fluorescein FITC
  • TRITC tetramethylrhodamine
  • 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.
  • 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.
  • 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
  • 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.
  • BHQ® black hole quencher
  • ATTO quencher e.g. ATTO 540Q, ATTO 580Q and ATTO 612Q
  • dimethylaminoazobenzenesulfonic acid dansyl
  • Iowa Black RQ Iowa Black FQ
  • IRDye QC-1 IRDye QC-1
  • QSY dyes e.g. QS
  • ATTO quenchers include, but are not limited to, ATTO 540Q, ATTO 580Q, and ATTO 612Q.
  • ATTO quenchers include, but are not limited to, ATTO 540Q, ATTO 580Q, and ATTO 612Q.
  • BHQ® Black Hole Quencher®
  • BHQ-3 BHQ-3
  • cleavage of the labeled detector ssDNA can be detected by measuring the colorimetric readout.
  • liberation of a fluorophore eg, liberation from a FRET pair, liberation from a quencher/fluor pair, etc.
  • liberation of a fluorophore can result in a wavelength shift (and thus color shift) of the detectable signal.
  • 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.
  • 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.
  • the viral vector may be a retroviral (retrovirus) vector, a lentiviral (lentivirus) vector, an adenoviral (adenovirus vector), or an adeno-associated viral vector.
  • AAV vaccinia viral vector
  • poxviral vector vaccinia virus vector
  • herpes simplex viral vector herpes simplex virus vector
  • the non-viral vector may be a plasmid, phage, naked DNA, DNA complex, mRNA (transcript) or PCR amplicon.
  • 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.
  • the phage may be selected from the group consisting of ⁇ gt4 ⁇ B, ⁇ -Charon, ⁇ z1, 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).
  • the promoter sequence may be a promoter that promotes transcription of RNA polymerase (eg, pol I, pol II, or pol III).
  • 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.
  • CMV cytomegalovirus
  • CBA chicken b-actin
  • RSV rous sarcoma virus
  • 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.
  • 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.
  • 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.
  • 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.
  • CRISPR/Cas system 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.
  • crRNA CRISPR RNA
  • gRNA guide RNA
  • 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).
  • PAM Protospacer Adjacent Motif
  • 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.
  • the Cas protein may correspond to Class 2.
  • the Cas protein corresponding to Class 2 may correspond to type V.
  • 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.
  • the isolated patient sample may include cell free DNA (cfDNA).
  • cfDNA cell free DNA
  • 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.
  • 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.
  • FIG. 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.
  • 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
  • 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 ⁇ -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 ⁇ -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).
  • 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 NanodropTM 2000 Spectrophotometer (Thermo-Fisher, Massachusetts, USA). The purified RNA was aliquoted and stored at -80 °C.
  • 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.
  • 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).
  • 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 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.
  • 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.
  • Fluorescent signals using collateral effects of LbCas12a were confirmed through a fluorophore-quencher reporter assay.
  • 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.
  • the FQ reporter 5 ⁇ /6-FAM/TTATT/BHQ1/3 ⁇
  • a 96 well black plate was prepared. plate), and the fluorescence signal was measured for 1 hour (FAM) at 1 minute intervals.
  • FluoroskanTM Microplate Fluorometer Thermo-Fisher, Massachusetts, U.S.A).
  • 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.
  • 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
  • the normal DNA-specific crRNA significantly cleaved the normal DNA, but the mutant DNA containing the 15bp deletion did not cut at all.
  • Example 2 CRISPR/Cas system designed to specifically amplify EGFR exon19 deletion mutant and confirmation of EGFR exon19 deletion mutant detection sensitivity
  • 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.
  • mutant DNA at a 1:100,000 dilution was not detected using conventional liquid biopsy.
  • 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).
  • 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 .
  • 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 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.
  • CRISPR/Cas12a amplification system 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.
  • LbCas12a/crRNA recognizes a target DNA
  • non-specific ssDNase which is a side effect of LbCas12a
  • 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.
  • 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. .
  • "Blank” indicates that only Cas12a/crRNA ribonucleic acid protein was cultured without DNA.
  • 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 CRISPR/Cas12a system can effectively increase a low fluorescence signal that is indistinguishable from a background signal.
  • exon 19 deletion mutant subtypes 1 to 4 represent forms in which 18 bp, 15 bp, 16+2 bp, and 9 bp of exon 19 are deleted, respectively.
  • FIGS. 9 to 12 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 .
  • P represents a positive sample previously confirmed by tissue biopsy
  • 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 clea
  • FIGS. 11 and 12 show 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).
  • 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.
  • 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.

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Abstract

La présente invention concerne un système CRISPR/Cas pouvant amplifier spécifiquement l'ADN mutant EGFR, ainsi que son utilisation pour le diagnostic du cancer. Un système d'amplification CRISPR/Cas12a peut être utilisé pour amplifier spécifiquement et efficacement l'ADN mutant issu d'un échantillon sanguin et confirmer ainsi les mutations présentant un ratio inférieur à 0,5 % pouvant être confondues avec des erreurs de séquençage.
PCT/KR2023/001357 2022-01-28 2023-01-30 Composition et procédé de détection d'acides nucléiques fondés sur un signal de fluorescence pouvant détecter spécifiquement une mutation egfr WO2023146370A1 (fr)

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