WO2019156475A1 - Mutant cell-free dna isolation kit and mutant cell-free dna isolation method using same - Google Patents

Abstract

The present invention relates to a mutant cell-free DNA isolation kit and a mutant cell-free DNA analysis method, both using a CRISPR/Cas system and an exonuclease.

Description

 Mutant Cell Free Gene Isolation Kit and Method for Isolating Mutant Cell Free Gene

The present invention relates to a mutant cell free DNA separation kit and a method for mutant cell free gene separation using the same. More specifically, the present invention relates to mutant cell free gene isolation kits and methods of isolation comprising a CRISPR / Cas system and exonuclease, for detecting mutant genes in trace cell free gene samples.

Recently, the importance of early diagnosis of cancer disease has been highlighted all over the world, and thus the importance of research on early cancer diagnosis methods is increasing. However, until now, the diagnosis of cancer mainly consists of invasive methods such as tissue sample collection and endoscopy. Existing methods are performed by extracting a part of the suspected area under a microscope, so that the patient feels less discomfort, scars remain, and recovery takes time.

Molecular diagnostics using liquid biopsy has attracted attention as an alternative to such invasive diagnostic and test methods. Because liquid biopsies use a non-invasive method, test results are faster and, unlike tissue samples that were able to analyze only a fraction of the disease, body fluids, or liquid biosamples, provide a multifaceted analysis of the disease. Can be done. In particular, liquid biopsies are expected to exert an excellent utility in the diagnosis of cancer, and it is possible to analyze cancer-derived DNA present in blood by body parts only by examining body fluids such as blood and urine for detailed observation of cancer occurrence and metastasis. It is predicted.

      Recently, studies on cell-free DNA (cf DNA) released from the tumor into the bloodstream and present in the blood in association with the liquid-biopsy have been actively conducted. As technology advances in the isolation and detection of cfDNAs from various biological samples such as blood, plasma or urine, liquid biopsies will become more effective and reliable tools for monitoring cancer risk patients. Cancer origin on cf DNA in more than 75% of patients with advanced pancreatic cancer, ovarian cancer, colon cancer, bladder cancer, gastric esophageal cancer, breast cancer, melanoma, hepatocellular carcinoma, head and neck cancer, and in more than 50% of kidney, prostate cancer or thyroid cancer patients The gene was identified. In the clinical diagnosis of metastatic colorectal cancer patients, the sensitivity of cf DNA to the detection of KRAS mutation was 87.2% and the specificity was 99.2%.

      These studies show the possibility of cancer diagnosis through cf DNA analysis, and cancer-derived genes in cf DNA are spotlighted as next generation biomarkers. By identifying tumor specific gene mutations from cf DNA from cancer patients, early stage tumors are being diagnosed. However, there are many limitations in the current technique for analyzing cfDNA in a liquid sample such as blood and urine and detecting a mutation occurring in a gene to diagnose cancer early (Patent Document 1). In particular, very low concentrations of cfDNA are present in samples of urine, cerebrospinal fluid, plasma, blood, or body fluids, and cfDNA may cause natural genetic mutations in the aging process. Most of the cf DNA in patient plasma is derived from normal somatic cells. Because of the wild type (wt) of the gene, current sequencing technology is almost impossible to accurately diagnose the presence of cancer cell-derived genes in the cf DNA. Therefore, in order to diagnose early stage cancer with trace amounts of cf DNA, it is necessary to remove normal genes and specifically amplify cancer cell-derived genes. Accordingly, there is an urgent need for a method for improving detection sensitivity and accurate early diagnosis of cancer.

       On the other hand, genome editing based on recently developed clustered regularly interspaced short palindrome repeats (RNA-guided CRISPR) -associated nuclease Cas proteins provides target knock-out, transcriptional activation and single guide RNA. It provides a breakthrough technique for inhibition with (sgRNA) (ie crRNA-tracrRNA fusion transcript), which demonstrated scalability by targeting numerous gene positions. The CRISPR-Cas system consists of guide RNA (gRNA) having a sequence complementary to the target gene or nucleic acid and CRISPR enzyme, a nuclease capable of cleaving the target gene or nucleic acid, and the gRNA and CRISPR enzyme form a CRISPR complex. And the target gene or nucleic acid is cleaved or modified by the formed CRISPR complex. The CRISPR system is an immune system of prokaryotes and archaea. Recently, research on the utility of the CRISPR system has been rapidly increasing (Non Patent Literature 1 and Non Patent Literature 2), but the sequence capture method and disease used in genome sequencing. No attempt was made to utilize the diagnosis.

Therefore, the present inventors have diligently tried to find a method for separating mutant genes on cfDNA, and as a result, the technique of specifically cleaving a small amount of cfDNA normal genes with the CRISPR / Cas system and amplifying the uncut mutant genes specifically By using the present invention was completed.

(Patent Document 1) Korean Unexamined Patent 10-2016-0129523

(Non-Patent Document 1) Jinek et al. Science, 2012, 337, 816-821

(Non-Patent Document 2) Zalatan et al. Cell, 2015, 160, 339-350

(Non-Patent Document 3) Tian J, Ma K, Saaem I., Mol Biosyst, 2009, 5 (7), 714-722

(Non-Patent Document 4 Michael, L .. Metzker, Nature Reviews Genetics, 2010, 11, 31-46

An object of the present invention is to provide a kit for mutant cell free gene isolation.

Another object of the present invention is to provide a mutant genotyping method.

The present invention provides a kit for isolating a mutant cell-derived gene comprising:

A composition for amplifying a wt cell free DNA and a mutant cell free DNA, comprising a primer protected at the 5 'end;

Said wild type cell free gene specific guide RNA;

Cas protein for cleaving said wild type cell free gene; And

Exonucleases to remove the wild type cell free gene.

The mutant cell free gene may be DNA comprising a cancer specific mutation.

 As used herein, the term “cell-free DNA (cfDNA)” refers to a gene derived from cancer cells that can be found in biological samples such as blood, plasma, or urine derived from cancer patients due to tumor cells. , Necrosis, apoptosis or activation in normal and / or cancer cells of the urinary tract and released into urine, blood and the like through various cellular physiological processes. Urine, cerebrospinal fluid (CSF), plasma, blood, or bodily fluids are readily available samples, and repetitive sampling enables the collection of large quantities of simple, non-invasive samples.

As used herein, the term “CRISPR / Cas system” consists of a guide RNA (gRNA) having a sequence complementary to a gene or nucleic acid and a CRISPR enzyme, a nuclease capable of cleaving a target gene or nucleic acid, and gRNA and CRISPR enzyme. Forms a CRISPR complex and cleaves or modifies the target gene or nucleic acid by the formed CRISPR complex.

Cas protein refers to an essential protein element in the CRISPR / Cas system, which forms an active endonuclease when complexed with two RNAs called CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). Form. Cas protein gene and protein information can be obtained from GenBank of the National Center for Biotechnology Information (NCBI), but is not limited thereto.

As used herein, the term “guide RNA” refers to RNA specific for a target DNA, which is expressed through transcription of a linear double-stranded DNA delivered into a cell to recognize a target gene sequence and form a complex with a Cas protein. It is RNA that is brought to the target DNA. The “target gene sequence” is a nucleotide sequence existing in a target gene or nucleic acid, specifically, a nucleotide sequence of a target region in the target gene or nucleic acid, wherein the “target region” is a guide nucleic acid-editor in the target gene or nucleic acid. It is a site that can be modified by protein.

As used herein, the term “PAM sequence” refers to a target sequence recognized by the CRISPR-Cas complex as a sequence of about 3bp to 6bp located next to a target sequence, and the CRISPR-Cas complex recognizes a PAM sequence. After that, the specific position is cut.

The kit may be for separating mutant cell free genes from wild type cell free genes and mutant cell free genes contained in liquid samples (blood, plasma or urine samples, etc.) isolated from suspected cancer.

The Cas protein is Streptococcus pyogenes Cas9 (SpCas9), Streptococcus thermophilus Cas9 (StCas9), Streptococcus pasteurianus (SpaCas9), Campylobacter jejuni Cas9 (CjCas9), Staphylococcus aureus (SaCas9as Cas9a 9), Francisella ), But may not be limited to Neisseria meningitis Cas9 (NmCas9) Prevotella or Francisella 1 (Cpf1).

The Cas protein or gene information may be obtained from a known database such as GenBank of the National Center for Biotechnology Information (NCBI), but is not limited thereto.

The guide RNA may be a dual RNA (single-chain RNA) or a sgRNA (sgRNA) including crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA).

The guide RNA may include two or more types of guide RNAs specific for a plurality of wild type cell free genes. That is, the kit of the present invention is a kit capable of multiplexing.

Such guide RNA production methods are well known in the art.

The primer protected by the 5 'end may be the 5' end of the primer protected by a phosphothioate bond.

The gene amplification composition may be a PCR composition.

 In the present invention, the term "amplification reaction" means a reaction for amplifying a target nucleic acid sequence, it can be carried out by polymerase chain reaction (PCR). The PCR may be reverse transcription polymerase chain reaction (RT-PCR), multiplex PCR, real-time PCR, assembly PCR, fusion PCR, ligase chain reaction ( Ligase chain reaction (LCR), but is not limited thereto.

As used herein, the term "primer" is one of single stranded oligonucleotides, which may also include ribonucleotides, preferably deoxyribonucleotides. The primers hybridize or anneal to one site of the template to form a double stranded structure. In the present invention, the primer may be hybridized or annealed to the NGS sequencing adapter sequence. Annealing refers to the placement of oligonucleotides or nucleic acids into a template nucleic acid, which condition causes the polymerase to polymerize the nucleotides to form a nucleic acid molecule that is complementary to the template nucleic acid or portion thereof. Hybridization means that two single-stranded nucleic acids form a duplex structure by pairing complementary sequences. The primer can serve as a starting point for synthesis under conditions that result in the synthesis of a primer extension product complementary to the template.

The PCR composition may include components well known in the art, in addition to the 5′-end protected primers specific for each of the wild type cell free gene and the mutant cell free gene. Specifically, it may include a PCR buffer (PCR buffer), dNTP, DNA polymerase (DNA Polymerase) and the like.

As used herein, the term “exonuclease” refers to an enzyme that sequentially degrades nucleotides from the 3′-end or 5′-end of a polynucleotide chain in a DNA sequence. 3'-5 'exonuclease is an enzyme that breaks down a phospho-diester bond at the 3'-end, and 5'-3' exonuclease is a phosphoester at the 5'-end. diester) An enzyme that breaks down bonds.

The exonuclease can be used any exonuclease known in the art. Specifically, the exonuclease may include 3 '→ 5' exonuclease and / or 5 '→ 3' exonuclease. Specifically, exonuclease III, exonuclease I, T5 exonuclease, T7 exonuclease, exonuclease T, exonuclease V, Lambda exonuclease, and exonuclease VII, and the like. Preferably, it may be, but is not necessarily limited to, exonuclease T7 and exonuclease T (single strand specific nuclease).

When a sample containing a wild type cell free gene and a mutant cell free gene is reacted with a gene amplification composition comprising a primer having a 5 'end protected, the wild type cell free gene and the mutant cell free gene ends are protected by phosphothioate. do. These protected DNAs are not degraded due to phosphothioate linkages even when treated with exonucleases.

When the DNA is cleaved by wild type cell free DNA by wild type cell free gene specific guide RNA and Cas protein, the unprotected nucleic acid ends are exposed to exonuclease, which degrades the wild type cell free gene DNA. Removed and only mutant cell free genes are isolated (FIG. 1).

In one embodiment of the present invention, when using the guideRNA targeting the KRAS gene, only wt KRAS satisfying the 5'-NGG-3 'PAM sequence was selectively cut by Cas9. At this time, by treating T7 Exonuclease and Exonuclease T, it was confirmed by electrophoresis that wt KRAS DNA cut by Cas9 was completely removed.

In another aspect, the present invention provides a mutant genotyping method comprising the following steps:

i) the wild type cell free gene and the mutant cell free gene in an isolated sample containing at least one wild type cell-free gene and at least one mutant cell free DNA. Amplifying using a primer protected at the end;

ii) treating only the amplified wild type cell free gene by treating guide RNA and Cas protein that specifically binds to the wild type cell free gene;

iii) removing only the truncated wild-type cell free gene by treating an mutant cell free gene and a sample containing the truncated cell free gene with an exonuclease;

iv) amplifying the mutant cell free gene remaining in the sample; And

v) analyzing the amplified mutant cell free genes.

I) The isolated sample containing at least one wild-type cell-free gene and at least one mutant cell free gene may be a liquid sample (blood, plasma or urine sample) isolated from a suspected cancer.

The mutant cell free gene may be DNA comprising a cancer specific mutation.

The mutant cell free gene analysis may be for providing information for diagnosis of cancer.

As used herein, the term “diagnosis” refers to determining the susceptibility of an object to a particular disease or condition, to determining whether an object currently has a particular disease or condition, or to a particular disease or condition. Determining the prognosis of one object at hand, or therametrics (eg, monitoring the condition of the object to provide information about treatment efficacy).

In the present invention, the diagnosis may include confirming whether the cancer develops and / or confirming the prognosis of the cancer.

The cancer may be early cancer.

Such cancers include, for example, squamous cell cancer (e.g., squamous cell cancer of the epithelium), small cell lung cancer, non-small cell lung cancer, lung cancer, peritoneal cancer, colon cancer, biliary tract tumors, nasopharyngeal cancer, laryngeal cancer, bronchial cancer , Oral cancer, osteosarcoma, gallbladder cancer, kidney cancer, leukemia, bladder cancer, melanoma, brain cancer, glioma, brain tumor, skin cancer, pancreatic cancer, breast cancer, liver cancer, bone marrow cancer, esophageal cancer, colon cancer, stomach cancer, cervical cancer, prostate cancer, ovarian cancer, Head and neck cancer and rectal cancer may be one or more selected from the group consisting of, but is not limited thereto.

The primer protected by the 5 'end may be the 5' end of the primer protected by a phosphothioate bond.

The Cas protein is Streptococcus pyogenes Cas9 (SpCas9), Streptococcus thermophilus Cas9 (StCas9), Streptococcus pasteurianus (SpaCas9), Campylobacter jejuni Cas9 (CjCas9), Staphylococcus aureus (SaCas9as Cas9a 9), Francisella ), But may not be limited to Neisseria meningitis Cas9 (NmCas9) Prevotella or Francisella 1 (Cpf1).

The guide RNA may be a dual RNA (single-chain RNA) or a sgRNA (sgRNA), including crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA).

The amplification may be performed by PCR, and PCR conditions may be set based on methods well known in the art.

The kit for mutant cell free gene isolation and mutant cell free gene isolation method of the present invention is a CRISPR / Cas system and exonuclease, and selects the normal somatic cell-derived gene (> 99.9%), which accounts for most of cfDNA in the blood. Can be removed with As a result, only mutant cell free genes (genes derived from cancer cells) can be analyzed on cfDNA, which can be useful for early cancer diagnosis.

       In addition, the kit for mutant cell free gene isolation and mutant cell free gene separation method of the present invention, by removing a gene derived from normal cells in the sample, a very small amount corresponding to the ratio of the mutant cell free gene (cancer cell derived gene) present in cfDNA Provides the effect of analyzing genes.

In addition, the present invention can use a plurality of normal gene-specific guideRNA, it is possible to simultaneously detect two or more oncogenic genes at the same time by a multiplexing system (multiplexing system).

1 is a schematic diagram of a process of amplifying only mtDNA (mutant cell free DNA) by selectively removing the wtDNA (wild type cell free DNA) in the sample in the present invention.

Figure 2 relates to the results of selective cleavage of wtDNA only in the sample using the CRISPR-Cas system according to the present invention.

Figure 3 relates to the results of the DNA cleaved by the CRISPR-Cas system according to the invention is selectively degraded by exonuclease.

Figure 4 relates to the results of the analysis of the sample removed KRAS wtDNA by treatment of Cas protein and exonuclease according to the present invention through the next generation sequencing.

Figure 5 relates to the results of analyzing the sample by removing the KRAS wtDNA by treatment with Cas protein and exonuclease according to the present invention through Sanger sequencing.

6 is a specific schematic diagram illustrating a process of amplifying several cancer-derived genes at once by treating Cas proteins and exonucleases according to the present invention and removing several target genes by multiplexing.

Figure 7 relates to the results proved through agarose gel electrophoresis that can only selectively remove the Mokpo gene (wt DNA) by using the Cas protein and exonuclease and multiplexing system according to the present invention.

Figure 8 relates to the results of the analysis of the next step sequence analysis of the samples treated with Cas protein and exonuclease according to the present invention and removed KRAS wtDNA and EGFR wtDNA using a multiplexing system.

9 is a schematic diagram showing a gene removal method grafted with a multiplexing system utilizing Cas9 ortholog in the present invention.

 The CRISPR / Cas system, which can selectively cleave target genes, is a highly accurate endonuclease, and Cas9 orthologes have different PAM region sequences. The PAM sequence is an essential gene sequence when the Cas9 protein recognizes a target gene. Even if the PAM sequence is not satisfied even if it binds with guide RNA, which is perfectly complementary to the target gene, the CRISPR Cas system does not work. In addition, if the guide RNA has a large number of target genes and non-complementary gene sequences, CRISPR / Cas may not recognize the target genes normally. In order to identify cancer genes generated for nucleotide substitution, guide RNAs complementary to wild-type genes were designed. When selecting the target of the guide RNA, the design was largely based on two criteria. If the nucleotide sequence before the substitution occurs in the PAM site, the guide RNA was designed based on the PAM site, and if it does not correspond to the PAM site, the guide RNA was added by adding a mismatch to the seed region. Was designed. The seed region of Cas9 is sensitive to the mismatch between the guide RNA and the target gene when the target gene is recognized. If there is a 1 to 2 nt mismatch in the seed region, the target gene is not clearly recognized.

       The guide RNA used in the present invention is designed to selectively recognize wild-type genes.

       In the present invention, when PCR amplification after cleavage of the target gene with Cas9, the wild type cell free DNA cut by Cas9 was less amplified than the mutant cell free DNA. However, a simple method of cutting wild-type DNA with Cas9 and PCR for mutant DNA did not reach the detection limit to identify trace mutant genes. Since the wild-type DNA fragment cleaved by Cas9 may serve as a primer in the PCR process, the wild-type-derived gene sequence may be included in the finally amplified PCR product. Even with the cleavage of Cas9's accuracy and selective cleavage of the wild-type gene, the truncated DNA fragments raised the background signal, making it impossible to reach the limit of detection for detecting trace amounts of cancer-derived genes (bottom left box in FIG. 1). See picture).

       However, in the present invention, by removing the truncated fragments of the wild-type DNA, which generate a background signal during the PCR process, it has sufficient detectability even for a small amount of the target gene. When amplifying the gene to be analyzed by PCR in the final process of trace cf DNA gene analysis, the 5 'end is amplified using a primer protected with a phosphothioate bond, or amplified PCR amplicon Ligation of the adapter (adatpor) containing a phosphothioate bond at both ends of the (ligation). If phosphodiester bonds linking nucleotides are replaced with phosphothioate bonds, the nuclease will not be able to cleave between the nucleotides in which the bond exists.

      When a target gene is protected at both ends by phosphothioate bonds, the exonuclease will not be cleaved by the exonuclease. However, when the target gene is cleaved by Cas9, new DNA ends not protected by phosphothioate are exposed to exonuclease, so that the DNA cut by Cas9 is removed by exonuclease.

      Therefore, the gene cutting and elimination techniques underlying the present invention play a complementary role, enabling a clear detection of cancer-derived genes in trace amounts of cf DNA that was previously impossible.

      In one embodiment of the present invention, it was confirmed that the isolated and purified SpCas9 protein and the guide RNA complex precisely cause the cleavage only at the gene target point that satisfies the PAM sequence of 5'-NGG-3 '.

      In the present invention, the mutation of the KRAS gene is one of the major mutations of the cancer gene, and is an important biomarker on cfDNA. In particular, the mutation of KRAS mainly occurs when the 35th nucleic acid on the cDNA is replaced with G by T, so that the 12th amino acid is replaced with valine from aspartic acid (asparatate) on the protein. The 35th guanosine (guanosine) is replaced with thymidine mtDNA is the base sequence corresponding to the PAM of Cas9 on the target gene is changed to NGT. SpCas9, which specifically recognizes NGG as a PAM sequence, cleaves DNA by specifically recognizing only wt KRAS among wt KRAS and mt KRAS (35G> T) (FIG. 2).

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Isolation and Purification of SpCas9

SpCas9 genetic information ((the entire sequence of the protein including His x 6, SEQ ID NO: 1) for protein expression of the recombinant purulent Streptococcus Cas9 (spCas9) containing a His x 6 tag at the N-terminus) The plasmid containing plasmid was transformed into BL21-DE3 species, which are sensitized cells, and treated with IPTG (Isopropyl bD-ThioGalactoside) in a liquid medium in which the transformed BL21-DE3 was growing. The cells were then incubated for 16 hours at 18 ° C. The cells were centrifuged at 5000 × g for 15 minutes, followed by NaH ₂ PO ₄ 50 mM (pH 8), NaCl 400 mM, imidazole 10 mM, PMSF 1 mM, DTT 1 mM, Triton X-100 1%, The cells were lysed with a lysis buffer containing 1 mg / ml of lysozyme, and the cells were crushed using an ultrasonic crusher, followed by centrifugation at 15000 × g for 30 minutes to separate the supernatant and cell debris. -Nitrilo Tri Set acid resin (Ni-NTA resin, Invitrogen, Carlsbad, CA) was added and after reaction for one hour at _{4 ℃, NaH 2 PO 4 50mM} (pH 8), NaCl 400mM, wash buffer of imidazole 20mM (wash buffer) The Ni-NTA resin was washed twice, and finally, spCas9 was added to Ni-NTA by adding elution buffer of NaH ₂ PO ₄ 50 mM (pH 8), NaCl 400 mM, and imidazole 250 mM. The buffer containing spCas9 was replaced with a buffer containing 20 mM HEPES (pH 7.5), 400 mM NaCl, 1 mM DTT, and 40% glycerol using an amicon ultra centrifugal filter.

Example 2. Synthesis of Guide RNA by In-vitro Transcription

DNA template containing T7 promoter and guide RNA information and T7 RNA polymerase were prepared in buffer containing 40 mM Tris-HCl (pH 7.9), 6 mM MgCl ₂ , 10 mM DTT, 10 mM NaCl, 2 mM spermidine, NTP and Rnase inhibitors. By reacting for 18 hours at ℃, guide RNA was synthesized in vitro (SEQ ID NOs: 2 and 3). The synthesized guide RNA was purified using an RNA purification kit.

Example 3. In vitro Cleavage

       500ng SpCas9 (hereinafter referred to as Cas9) isolated and purified according to Example 1 and 100ng guide RNA prepared according to Example 2 were combined at 37 ° C. for 5 minutes, Potassum acetate 50 mM (pH 7.9), Tris-acetate In a buffer consisting of 20 mM, Magnesium acetate 10 mM, and 1 mM DTT, 150 ng double-stranded DNA (dsDNA) was reacted with wild type KRAS gene (SEQ ID NO: 4) and mutant KRAS gene (KRAS D12V, SEQ ID NO: 6) at 37 ° C for 60 minutes. KRAS gene and amino acid sequence information is shown in Table 1 below.

TABLE 1

DNA fragments cleaved by Cas9 were confirmed by electrophoresis on 1.5% agarose gel and stained with EtBr. As a result, the isolated purified Cas9 protein and the guide RNA complex were confirmed to precisely cut only the gene target point satisfying the PAM sequence of 5'-NGG-3 '(see Fig. 2).

      One of the major mutations in cancer genes, the KRAS gene, is an important biomarker that can be detected on cfDNA. In particular, the variation of KRAS is important in that the 35th nucleic acid on cDNA is replaced with T by G, so that the 12th amino acid in KRAS protein is replaced by glycine (G) with valine (V) (SEQ ID NO: 5 and sequence). Number 7). In this way, the mtDNA in which the 35th guanosine is substituted with thymidine changes the nucleotide sequence corresponding to the PAM of Cas9 to NGT on the target gene. Cas9, which specifically recognizes NGG as a PAM sequence, cleaves DNA by specifically recognizing only wt KRAS among wt KRAS and mt KRAS (35G> T) (SEQ ID NO: 8).

   The guide RNA has a target sequence corresponding to position 1572nt on 2787nt DNA containing the wild type KRAS gene (SEQ ID NO: 4). Cas9 coupled with guide RNA cleaved the KRAS gene on 2787nt of DNA to make 1572nt long fragment and 1215nt short fragment in the electrophoresis results (see top of FIG. 2b and top of FIG. 3b). However, even when using the guide RNA, it was confirmed that the mtKRAS gene whose PAM sequence was changed to 5'-NGT-3 'was not cleaved by Cas9 (see FIG. 2b bottom and 3b bottom).

Example 4 Selective Removal of Wild-type Cell Free DNA Only

       Experiments were conducted to confirm that wild-type cell free genes were selectively cleaved and that such cleavage was selectively progressed by exonuclease.

       Target genes (wild type cell free DNA and cancer specific cell derived DNA isolated from cancer patient blood) were identified using primers protected at 5 ′ ends using phosphothioate binding (Table 2) and phusion polymerase. PCR amplification. After purifying wild type DNA (wtDNA) and mutant DNA (mtDNA) using a PCR purification kit (Quiagen Cat ID: 28104), the wtDNA and mtDNA were 90:10 (wtDNA ratio 90%; mtDNA ratio 10%), 99 : 1 (wtDNA ratio 99%; mtDNA ratio 1%), 99.9: 0.1 (wtDNA ratio 99.9%; mtDNA ratio 0.1%), 99.99: 0.01 (wtDNA ratio 99.99%; mtDNA ratio 0.01%), 99.999: 0.001 (wtDNA ratio A DNA sample was prepared by mixing at a ratio of 99.999%; mtDNA ratio 0.001%). 500 ng SpCas9 and 100 ng guide RNA were combined at 37 ° C. for 5 minutes, Potassium acetate 50 mM (pH 7.9), Tris-acetate 20 mM, Magnesium acetate 10 mM, DiThioThreitol , Reducing agent) was reacted with 100ng double strand DNA (dsDNA) purified in a buffer consisting of 1mM for 60 minutes at 37 ℃.

TABLE 2

After the reaction, exonuclease T7 and exonuclease T were added to carry out additional reaction at 37 ° C. for 60 minutes. DNA fragments treated with Cas9 and exonuclease were identified by electrophoresis on a 1.5% agarose gel, or sequenced by Sanger sequencing and next generation sequencing by PCR amplification.

       2787nt DNA containing the KRAS gene is protected at both ends by phosphothioate bonds (see FIG. 3A step 1). DNA whose both ends are protected by phosphothioate bonds is not degraded by treatment with exonuclease. However, when the DNA was cleaved by cas9, the unprotected nucleic acid ends were exposed to exonuclease, which resulted in the degradation of the DNA (see FIG. 3A steps 2 and 3). When using guide RNAs targeting the KRAS gene, only wt KRAS genes that satisfy the 5'-NGG-3 'PAM sequence are selectively truncated by Cas9. At this time, it was confirmed by electrophoresis that the wt KRAS DNA cut by Cas9 was completely removed by treating sonuclease T7 and sonuclease T (see FIG. 3b top right line). However, it was confirmed that mt KRAS DNA not cut by Cas9 was protected and decomposed due to sportioate binding at both ends, even though sonuclease was treated (see the rightmost bottom line in FIG. 3b).

Example 5. Confirmation of detection limit for target DNA

       When only the target DNA was selectively removed, the gene present in the trace was confirmed by the sequencing result. At this time, in order to confirm the detection limit for the trace gene, the experiment was performed by mixing wt DNA and mt DNA in different ratios. 90% (wtDNA ratio 90%; mtDNA ratio 10%), 99: 1 (wtDNA ratio 99%; mtDNA ratio 1%), 99.9: 0.1 wtDNA ratio 99.9%; mtDNA ratio 0.1%), 99.99: 0.01 (wtDNA ratio 99.99%; mtDNA ratio 0.01%), 99.999: 0.001 (wtDNA ratio 99.999%; mtDNA ratio 0.001%), mix with Cas and exonuclease After processing, sequencing was performed on the finally obtained sample (see FIG. 4).

      As shown in FIG. 4, it was confirmed in the NGS sequencing results that the ratio of mt DNA also increased in the Cas9-only sample. However, if the percentage of mtDNA in the initial analysis sample falls below 1%, the rate of mtDNA finally detected is significantly lowered. Considering that the ratio of mt DNA in cfDNA is generally 0.01% or less, it is not easy to confirm the result of the presence or absence of mtDNA when only the target gene is cleaved with Cas9. Therefore, the inventors carried out the confirmation of the detection limit of the target gene as follows.

      When the samples treated with Cas9 and exonuclease were analyzed by Sanger sequencing, it was confirmed that the portion corresponding to KRAS c.35 guanosine was read as thymine. When only Cas9 was treated, the histograms of guanosine and thymine were observed simultaneously in the portion corresponding to the 35th nucleic acid in the sample (NGS result mtDNA: 44%) where the ratio of the first mtDNA was 1%, and the ratio of mtDNA was 1 Sequences of mtDNA could not be observed in samples below% (see Cas_treatment on the left in FIG. 5). However, even in a sample in which the ratio of mtDNA was 0.01% when Cas9, exonuclease T, and exonuclease T7 were treated, the mutant sequence was confirmed on Sanger sequencing results (see FIG. 5 right, Cas + ExoT + ExoT7_treatment).

       As shown in FIG. 5, in the present invention, in the case of a sample treated with Cas9, exonuclease T7, and exonuclease T, the presence of mtDNA was present in the NGS result even in a sample having an initial ratio of mtDNA of 0.01% or less. Was clearly confirmed (the ratio of mtDNA of the sample detectable using only Cas9 was 1%). Therefore, in the present invention, the detection limit was extended to at least 100 times as compared to the method of cleaving the target gene using only the Cas9 system.

Example 6 Removal of Various Target DNA Using Multiplex System

       According to the following method, the removal of various target DNAs using the multiplex system was confirmed. EGFR sequences are shown in Table 3 below.

TABLE 3

PCR amplification of several target genes was performed using primers (Table 2) protected by 5 'terminus by phosphothioate linkages and phusion polymerase. A target DNA sample was prepared by mixing 1: 1 purified KRAS DNA and EGFR DNA using a PCR purification kit. 50 ng of 500 ng Cas9 and guide RNA (SEQ ID NO: 3) targeting KRAS and EGFR were mixed for 5 min. Subsequently, 100 ng target DNA samples purified in a buffer consisting of 50 mM of Potassium acetate (pH 7.9), 20 mM of Tris-acetate, 10 mM of Magnesium acetate, and 1 mM of DTT were reacted at 37 ° C. for 60 minutes.

    After reacting with Cas9, exonuclease T7 and exonuclease T were added to react 37 [deg.] C for 60 minutes. DNA fragments treated with Cas9 and exonuclease were identified by electrophoresis on a 1.5% agarose gel or by PCR amplification to confirm sequencing by Sanger sequencing and next-generation sequencing.

When a guide RNA targeting KRAS DNA and a guide RNA targeting EGFR DNA were mixed and combined with Cas9, the DNA in which wt KRAS DNA (2787nt) and wt EGFR DNA (2813nt) were mixed in a 1: 1 ratio was cut. Each target gene was confirmed to be truncated by the corresponding guide RNA, and electrophoresis confirmed that only the truncated gene was completely degraded by exonuclease (see FIG. 7B). On the contrary, it was confirmed that the DNA sample mixed with mt KRAS and mt EGFR in a 1: 1 ratio was not cleaved by Cas9 and was not degraded by exonuclease (see bottom of FIG. 7B).

        As described above, it was confirmed by electrophoresis that by applying a multiplexing system using a guide RNA targeting different genes together, it is possible to remove several target genes at the same time. By removing several genes simultaneously, several trace genes were identified at once on the NGS results.

Example 7 Identification of Limits of Detection for Various Target DNAs Using Multiplexing Systems

       The detection limit of the multiplexing system was confirmed by NGS analysis using a DNA sample in which wt DNA and mt DNA corresponding to KRAS gene and EGFR gene were mixed at different ratios. 90% (KRAS mtDNA ratio: 10%, EGFR mtDNA ratio: 10%, wt KRAS DNA ratio and 90% wt KRAS DNA, wt EGFR DNA, mt KEG DNA and mt EGFR DNA protected at both ends by phosphothioate bonds) wt EGFR DNA ratio 90%), 99: 1 (KRAS mtDNA ratio: 1%, EGFR mtDNA ratio: 1%, wt KRAS DNA ratio and wt EGFR DNA ratio respectively 99%), 99.9: 0.1 (KRAS mtDNA ratio : 0.1%, EGFR mtDNA ratio: 0.1%, wt KRAS DNA ratio and wt EGFR DNA ratio are 99.9% respectively, 99.99: 0.01 (KRAS mtDNA ratio: 0.01%, EGFR mtDNA ratio: 0.01%, wt KRAS DNA ratio and wt EGFR DNA ratio is 99.99%), 99.999: 0.001 (KRAS mtDNA ratio: 0.001%, EGFR mtDNA ratio: 0.001%, wt KRAS DNA ratio and wt EGFR DNA ratio are respectively 99.999%) The final sample obtained was sequenced after the aze treatment.

As with experiments using only guide RNAs corresponding to KRAS (tests on single target DNA), when the multiplexing system was used, only mutations were detected in the samples containing less than 1% of mt DNA using Cas9 alone. However, when Cas9 and exonuclease were treated together, it was confirmed that mtDNA was effectively detected even in the multiplexing system (see FIGS. 8 and 9).

9 is as follows. Cas9 orthologs recognize PAMs of different sequences. In the case of spCas9, only a gene having a sequence of 5′-NGG-3 ′ adjacent to the target gene 3 ′ can be cut, and the corresponding NGG sequence on the gene is called the PAM sequence of sp Cas9. PAM sequences of nmCas9, saCas9, cjCas9, AsCpf1, and FnCpf1 are 5'-NNNGMTT-3 ', 5'-NNGRRT-3', 5'-NNNVRYAC-3 ', 5'-TTTN-3', 5'-KYTV Perceived as -3 '. This different kind of Cas9 ortholog has a PAM sequence and must meet the PAM sequence, but can function normally. Multiplexing the orthologs of Cas9 can overcome the limitations of the diversity of targetable genes resulting from constraints of PAM sequences. Genetic diagnostics using various Cas9 orthologs, as shown in the diagram, can detect various types of cancer mutations beyond the limits of target genes with spCsa9.

As mentioned above, the specific part of the content of the present invention has been described in detail. For those skilled in the art, this specific technology is merely a preferred embodiment, and it is obvious that the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (13)

1. A composition for amplifying a wild type cell free gene and a mutant cell free DNA, comprising a primer protected at the 5 'end;

   Said wild type cell free gene specific guide RNA;

   Cas protein for cleaving said wild type cell free gene; And

   Exonucleases to remove the wild type cell free gene;

   Including, mutant cell free gene separation kit.
2. The method of claim 1,

   The mutant cell free gene is DNA comprising a cancer specific mutation,

   The kit is for separating mutant cell free genes from wild type cell free genes and mutant cell free genes contained in blood, plasma or urine samples isolated from cancer suspected subjects.

   Kit for mutant cell free gene isolation.
3. The method of claim 1,

   The Cas protein is Streptococcus pyogenes Cas9 (SpCas9), Streptococcus thermophilus Cas9 (StCas9), Streptococcus pasteurianus (SpaCas9), Campylobacter jejuni Cas9 (CjCas9), Staphylococcus aureus (SaCas9as Cas9a 9), Francisella ), Kit for isolating mutant cell free genes, which is Neisseria meningitis Cas9 (NmCas9) Prevotella or Francisella 1 (Cpf1).
4. The method of claim 1,

   The guide RNA is a mutant cell free gene separation kit is a dual RNA (dualRNA) or sgRNA (single-chain RNA) comprising crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA).
5. The method of claim 1,

   The guide RNA is a kit for isolating mutant cell free genes comprising two or more types of guide RNAs specific for a plurality of wild type cell free genes.
6. The method of claim 1,

   The 5 'end of the protected primer is a 5' end of the primer is protected by phosphothioate bond, the gene amplification composition is a PCR composition, mutant cell free gene separation kit.
7. i) the wild type cell free gene and the mutant cell free gene in an isolated sample containing at least one wild type cell free DNA and at least one mutant cell free DNA. Amplifying using a primer protected at the end;

   ii) treating only the amplified wild type cell free gene by treating guide RNA and Cas protein that specifically binds to the wild type cell free gene;

   iii) removing only the truncated wild-type cell free gene by treating an mutant cell free gene and a sample containing the truncated cell free gene with an exonuclease;

   iv) amplifying the mutant cell free gene remaining in the sample; And

   v) analyzing the amplified mutant cell free genes;

   Including, mutant genotyping method.
8. The method of claim 7, wherein

   I) The isolated sample comprising at least one wild type cell free gene and at least one mutant cell free gene is a blood, plasma or urine sample isolated from a suspected cancer.
9. The method of claim 7, wherein

   The mutant cell free gene is DNA comprising a cancer specific mutation,

   The mutant cell free gene analysis is to provide information for diagnosis of cancer, mutant genotyping method.
10. The method of claim 7, wherein

    The 5 'end protected primer is a mutation genotyping method, wherein the 5' end of the primer is protected by phosphothioate bonds.
11. The method of claim 7, wherein

    The Cas protein is Streptococcus pyogenes Cas9 (SpCas9), Streptococcus thermophilus Cas9 (StCas9), Streptococcus pasteurianus (SpaCas9), Campylobacter jejuni Cas9 (CjCas9), Staphylococcus aureus (SaCas9as Cas9a 9), Francisella ), Neisseria meningitis Cas9 (NmCas9) Prevotella or Francisella 1 (Cpf1).
12. The method of claim 7, wherein

    Wherein said guide RNA is a dual RNA (dualRNA) or sgRNA (single-chain RNA) comprising crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA).
13. The method of claim 7, wherein

     Wherein said amplification is carried out by PCR.

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