An ultrasensitive target nucleic acid enrichment detection method based on programmable nuclease

Abstract

The present invention discloses an ultra-sensitive target nucleic acid enrichment detection method based on programmable nuclease, which belongs to the field of biotechnology. The enrichment method of the present invention is based on the specific cutting of programmable nuclease, and through the design and optimization of sgRNA, guide DNA or guide RNA, sgRNA, guide DNA or guide RNA and Cas protein or Ago protein form a ribonucleoprotein complex (ribonucleoprotein), which can continuously and specifically remove non-target nucleic acids while amplifying at a constant temperature, so that the number of target nucleic acids in the sample increases continuously and exponentially, thereby enriching or detecting target nucleic acids such as low-abundance mutant genes or methylated DNA.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an ultra-sensitive target nucleic acid enrichment detection method based on programmable nuclease. Background Art

[0002] Somatic mutations are closely related to tumorigenesis, and mutation detection is of great significance for early disease screening, precise treatment decisions, and recurrence monitoring. In recent years, non-invasive diagnosis represented by liquid biopsy has become a trend. Liquid biopsy analyzes the genotype of tumors by detecting free nucleic acid fragments derived from tumors in body fluids. It has the advantages of being non-invasive and easy to implement dynamic monitoring. However, liquid biopsy faces the following problems: 1) Mutated nucleic acids have high sequence homology with wild-type nucleic acids, usually with only a single or a few nucleotide differences, which poses a challenge to identifying low-abundance mutant alleles in a large number of free wild-type alleles; 2) The detection of low-abundance methylated genes in body fluids also faces the same problem. The methylated genes have the same sequence as the non-methylated genes, and only the base modifications are different, which poses a challenge to identifying low-abundance methylated genes in a large number of free non-methylated genes.

[0003] At present, highly sensitive detection of mutant alleles usually uses next-generation sequencing (NGS) or brute force counting relying on digital PCR to detect weak signals. Especially in the detection of minimal residual disease (MRD), for extremely low-abundance mutant genes, it is mainly based on ultra-deep (50000X) NGS sequencing, which is expensive, time-consuming, and difficult to popularize in clinical practice. Therefore, it is urgent to develop a low-cost ultra-sensitive method for detecting extremely low-abundance mutant alleles to promote clinical application.

[0004] In recent years, diagnostic technology based on CRISPR-Cas programmable nucleases has become a research hotspot. The CRISPR-Cas system is an adaptive immune defense system formed by archaea and most bacteria to resist viral invasion during biological evolution. It consists of Cas effector proteins and guide RNAs. Under the guidance of guide RNAs, Cas proteins recognize and cut target sequences containing 5' PAM sites. The low-abundance mutant allele detection method based on CRISPR-Cas aims to enable Cas proteins to specifically cut wild-type alleles, remove a large amount of wild-type nucleic acids, and retain mutant alleles through special guide RNA design to improve the sensitivity of downstream analysis. At the same time, some researchers have established similar low-abundance mutant allele detection methods based on Argonaute protein (Ago) by forming a complex with guideDNA.

[0005] The cleavage of Cas proteins is restricted by PAM sites. When mutations lead to the destruction of PAM sites, in the same sample, Cas proteins target and cleave a large number of wild-type nucleic acids while retaining mutant nucleic acids. Researchers have developed low-abundance mutant allele detection methods such as DASH (Depletion of Abundant Sequences by Hybridization) and CUT-PCR for mutations located at PAM sites. Argonaute proteins are not restricted by PAM sites, and researchers have developed NAVIGATER (Nucleic Acid enrichment Via DNA Guided Argonaute from Thermus thermophilus) low-abundance mutant allele detection methods.

[0006] Although the DASH method and CUT-PCR have improved the sensitivity of downstream analysis to a certain extent, the effectiveness of existing programmable endonuclease-based detection methods is affected by ineffective binding events between the enzyme and the target. Since the dissociation efficiency of cas9 is very slow, some wild-type allele targets are protected from being cut, and non-specific off-target cutting will deplete extremely rare mutant alleles. NAVIGATER also has similar problems. To overcome these shortcomings, researchers used multiple rounds of selective cutting of wild-type alleles, followed by polymerase chain reaction, to enrich the fragments of mutant alleles by 10 times. Despite these improvements, the high sensitivity of mutant allele detection still requires the use of next generation sequencing (NGS) or relies on digital PCR, making these methods laborious, time-consuming and expensive.

[0007] For low-abundance methylated gene detection in liquid biopsy, the above technologies have similar problems and are difficult to meet the requirements. Summary of the invention

[0008] The purpose of the present invention is to provide an ultra-sensitive tumor-related gene enrichment method based on programmable nucleases. The method of the present invention can efficiently enrich low-abundance mutant genes and methylated genes, and the microfluidic chip technology is used to automate the method of the present invention.

[0009] In order to achieve the above-mentioned invention object, the following technical scheme is adopted:

[0010] A ribonucleoprotein complex comprising:

[0011] Component (a) comprises a nucleic acid region capable of complementary pairing with a target nucleic acid and a 0-4 bp mismatch fragment; or a nucleic acid region capable of complementary pairing with a target nucleic acid and an adjacent PAM including a mutation-prone site;

[0012] Component (b), capable of binding to the target nucleic acid and breaking the target nucleic acid strand;

[0013] Wherein, component (a) is selected from tracrRNA and / or crRNA and / or sgRNA and / or tracrRNA derivatives and / or crRNA derivatives and / or sgRNA derivatives and / or guide DNA and / or guide RNA; component (b) includes Cas protein and / or Cas protein derivatives and / or Ago protein and / or Ago protein derivatives; components (a) and (b) can be combined. Preferably, component (a) can carry a label, and the label includes streptavidin or biotin.

[0014] Preferably, the length of the mismatched nucleotide fragment is 0 bp, 1 bp, 2 bp, 3 bp or 4 bp.

[0015] Preferably, component (a) carries a chemical modification comprising a sulfur-substituted oxygen or a methoxy modification.

[0016] Preferably, the sgRNA, guide DNA or guide RNA in component (a) includes mismatched nucleotide fragments. The mismatched nucleotide fragments designed in the present invention are conducive to expanding the scope of use of sgRNA, guide DNA or guide RNA, and enable sgRNA, guide DNA or guide RNA to recognize more mutations.

[0017] Preferably, the PAM adjacent to the sgRNA in component (a) contains a tumor hotspot mutation site.

[0018] Preferably, the spacer sequence length of the sgRNA in component (a) is 16-22 nt.

[0019] More preferably, the sgRNA sequence is as follows:

[0020] UCUUAAUUCCUUGAUAGCGA (SEQ ID NO.1);

[0021] UAGCUACAGUGAACUCUCGA (SEQ ID NO.2);

[0022] UAGCUACAGUGAAAUCACGA (SEQ ID NO.3);

[0023] GCUACAGUGAACUCUCGA (SEQ ID NO.4);

[0024] GUCUAGCUACAGUGAAA (SEQ ID NO. 5);

[0025] GUCUAGCUGCAGUGAAA (SEQ ID NO. 6);

[0026] GGCAGCCGAAGGGCAUGAGC (SEQ ID NO. 27);

[0027] GGCAGCCGAAGAGCAUGAGC (SEQ ID NO. 28);

[0028] AAUUUUUGUUUGAGUGGUUG (SEQ ID NO. 37);

[0029] UCCAGCUGUAUCCAGUAUGU (SEQ ID NO. 42).

[0030] More preferably, the guide DNA sequence is as follows:

[0031] Forward guide: p-TAGATTTCACTGTAGC-3' (SEQ ID NO. 43);

[0032] Reverse guide: p-TTCTAGCTACAGTGAA -3' (SEQ ID NO. 44).

[0033] Preferably, component (b) includes at least one of the following: Cas12a, SacCas9, CjCas9, SpCas9, NmCas9, Sp-Cas9 HF1, evoCas9, HypaCas9, HiFi Cas9, Sniper-Cas9, xCas9, eSpCas9 1.1, SuperFiCas9, SaCas9, SaCas9-HF, efSaCas9, ScCas9, Cas9—Sc++, Cas9 HiFi-Sc++, SpaCas9, SpaCas9-HF, FnCas9, AnaCas9, SpyCas9, FnCas12a, LbCas12a, AsCas12a, CbAgo, TtAgo, PfAgo, KmAgo or KpAgo.

[0034] More preferably, Cas12a is from at least one of the following species: Francisella novicida ; Acidaminococcus ; Lachnospiraceae .

[0035] More preferably, SacCas9 is from a bacterial species Staphylococcus aureus .

[0036] More preferably, CjCas9 is from a bacterial species Campylobacter jejuni .

[0037] More preferably, SpCas9 is derived from a bacterial species Streptococcus pyogenes .

[0038] More preferably, NmCas9 is derived from a bacterial species Neisseria meningitidis .

[0039] More preferably, SaCas9 is derived from a bacterial species Staphylococcus aureus.

[0040] More preferably, ScCas9 is derived from a bacterial species Streptococcus canis.

[0041] More preferably, SpaCas9 is derived from a bacterial species Streptococcus pasteurianus.

[0042] Preferably, in the ribonucleoprotein complex, the final concentration of component (a) is 0.1-2 μM.

[0043] Preferably, in the ribonucleoprotein complex, the final concentration of component (b) is 0.1-3 μM.

[0044] Preferably, the ribonucleoprotein complex further comprises a hydrogen ion buffer.

[0045] More preferably, in the ribonucleoprotein complex, the final concentration of the hydrogen ion buffer is 1 mM-2 M.

[0046] The invention also discloses the use of the ribonucleoprotein in enriching and / or detecting mutant genes.

[0047] The invention also discloses the use of the ribonucleoprotein in enriching and / or detecting methylated DNA.

[0048] Preferably, the genes include at least one of the following: EGFR, BRAF, PIK3CA, TP53, LRP1B, APC, CYP1A1, NP01, EPHX1, KRAS, BRCA1, BRCA2, MET, MLH1, MSH2, MSH3, MSH6, PALB2, BMPR1A, SMAD4, STK11, PTEN, AXIN2, BLM, BUB1B, CDH1, CEP57, CHEK2, ENG, EPCAM, FLCN, GALNTI2, GREM1, FAT4, KMT2D, KMT2C, ARID1A, FAT1, PTEN, ATM, ZFHX3, CREBBP, GRIN2A, NRAS or NF1.

[0049] The present invention also discloses a method for preparing a ribonucleoprotein complex, comprising: mixing and incubating Cas9, sgRNA and a hydrogen ion buffer, and mixing and incubating Ago, guide DNA or RNA and a hydrogen ion buffer.

[0050] Preferably, the hydrogen ion buffer comprises HEPES.

[0051] Preferably, in the preparation of the ribonucleoprotein complex, the final concentration of Cas9 used is 0.1-3 μM.

[0052] More preferably, in the preparation of the ribonucleoprotein complex, the final concentration of Cas9 used is 2.5 μM.

[0053] Preferably, in the preparation of the ribonucleoprotein complex, the final concentration of the sgRNA used is 0.1-2 μM.

[0054] More preferably, in the preparation of the ribonucleoprotein complex, the final concentration of the sgRNA used is 2.5 μM.

[0055] Preferably, in the preparation of the ribonucleoprotein complex, the final concentration of HEPES used is 1 mM-2M.

[0056] Preferably, in the preparation of the ribonucleoprotein complex, the final concentration of HEPES used is 1M.

[0057] Preferably, in the preparation of the ribonucleoprotein complex, the reaction temperature is 34-39° C. and the reaction time is 3-18 min.

[0058] More preferably, in the preparation of the ribonucleoprotein complex, the reaction temperature is 37° C. and the reaction time is 10 min.

[0059] The present invention also discloses a reaction system for enriching mutant genes of programmable enzymes and / or enriching methylated DNA, and the reaction system comprises the above-mentioned ribonucleoprotein complex.

[0060] Preferably, the ribonucleoprotein complex comprises:

[0061] Component (a), comprising a nucleic acid region capable of complementary pairing with a target nucleic acid and a 0-4 bp mismatch fragment;

[0062] Component (b), capable of binding to the target nucleic acid and breaking the target nucleic acid strand;

[0063] Wherein, component (a) is selected from tracrRNA and / or crRNA and / or sgRNA and / or tracrRNA derivatives and / or crRNA derivatives and / or sgRNA derivatives and / or guide DNA and / or guide RNA; component (b) includes Cas protein and / or Cas protein derivatives and / or Ago protein and / or Ago protein derivatives; components (a) and (b) can be combined. Preferably, component (a) carries a label, and the label includes streptavidin or biotin.

[0064] Preferably, the length of the mismatched nucleotide fragment is 0 bp, 1 bp, 2 bp, 3 bp or 4 bp.

[0065] More preferably, component (a) carries a chemical modification comprising a sulfur-substituted oxygen or a methoxy modification.

[0066] More preferably, the sgRNA, guide DNA or guide RNA in component (a) includes mismatched nucleotide fragments.

[0067] More preferably, the spacer sequence length of the sgRNA, guide DNA or guide RNA in component (a) is 16-22 nt.

[0068] More preferably, the sgRNA sequence is as follows:

[0069] UCUUAAUUCCUUGAUAGCGA (SEQ ID NO.1);

[0070] UAGCUACAGUGAACUCUCGA (SEQ ID NO.2);

[0071] UAGCUACAGUGAAAUCACGA (SEQ ID NO.3);

[0072] GCUACAGUGAACUCUCGA (SEQ ID NO.4);

[0073] GUCUAGCUACAGUGAAA (SEQ ID NO. 5);

[0074] GUCUAGCUGCAGUGAAA (SEQ ID NO. 6);

[0075] GGCAGCCGAAGGGCAUGAGC (SEQ ID NO. 27);

[0076] GGCAGCCGAAGAGCAUGAGC (SEQ ID NO. 28);

[0077] AAUUUUUGUUUGAGUGGUUG (SEQ ID NO. 37);

[0078] UCCAGCUGUAUCCAGUAUGU (SEQ ID NO. 42).

[0079] More preferably, the guide DNA sequence is as follows:

[0080] Forward guide: p-TAGATTTCACTGTAGC-3' (SEQ ID NO. 43);

[0081] Reverse guide: p-TTCTAGCTACAGTGAA -3' (SEQ ID NO. 44).

[0082] More preferably, component (b) includes at least one of the following: Cas12a, SacCas9, CjCas9, SpCas9, NmCas9, Sp-Cas9 HF1, evoCas9, HypaCas9, HiFi Cas9, Sniper-Cas9, xCas9, eSpCas9 1.1, SuperFiCas9, SaCas9, SaCas9-HF, efSaCas9, ScCas9, Cas9—Sc++, Cas9 HiFi-Sc++, SpaCas9, SpaCas9-HF, FnCas9, AnaCas9, SpyCas9, FnCas12a, LbCas12a, AsCas12a, CbAgo, TtAgo, PfAgo, KmAgo or KpAgo.

[0083] More preferably, Cas12a is from at least one of the following strains: Francisella novicida ; Acidaminococcus ; Lachnospiraceae .

[0084] More preferably, SacCas9 is from a bacterial species Staphylococcus aureus .

[0085] More preferably, CjCas9 is from a bacterial species Campylobacter jejuni .

[0086] More preferably, SpCas9 is derived from a bacterial species Streptococcus pyogenes .

[0087] More preferably, NmCas9 is from a bacterial species Neisseria meningitidis .

[0088] More preferably, SaCas9 is derived from a bacterial species Staphylococcus aureus.

[0089] More preferably, ScCas9 is derived from a bacterial species Streptococcus canis.

[0090] More preferably, SpaCas9 is derived from a bacterial species Streptococcus pasteurianus.

[0091] More preferably, in the ribonucleoprotein complex, the final concentration of component (a) is 0.1-2 μM.

[0092] More preferably, in the ribonucleoprotein complex, the final concentration of component (b) is 0.1-3 μM.

[0093] More preferably, the ribonucleoprotein complex further comprises a hydrogen ion buffer.

[0094] More preferably, in the ribonucleoprotein complex, the final concentration of the hydrogen ion buffer is 1 mM-2 M.

[0095] Preferably, in the reaction system, the final concentration of the ribonucleoprotein complex is 0.1-2 μM.

[0096] Preferably, the reaction system further comprises: primers, target gene, enzyme and auxochromogen.

[0097] Preferably, the reaction system further comprises: dNTP and single-stranded binding protein.

[0098] More preferably, the target gene includes: genomic DNA or cell-free DNA.

[0099] More preferably, the target gene comprises genomic DNA extracted from at least one of the following cell lines:

[0100] EGFR 19del wild-type cell line; EGFR 19 E746_A750 del (2235-2249del) cell line; EGFR19 E746_A750 del (2236-2250del); BRAF V600E mutant; BRAFV600E wild-type cell line; B-CPAP cell line.

[0101] More preferably, the primer comprises a nucleotide sequence capable of binding to the gene of interest and directing synthesis.

[0102] More preferably, the primer comprises at least one of the following sequences:

[0103] GCATGTGGCACCATCTCACA (SEQ ID NO. 15);

[0104] AGAGCAGCTGCCAGACATGA (SEQ ID NO. 16);

[0105] CTACACCTCAGATATATTTC(SEQ ID NO.19);

[0106] TGGATCCAGACAACTGT(SEQ ID NO.20);

[0107] TACGTGATGGCCAGCGTGGA (SEQ ID NO. 23);

[0108] ACTGGGAGCCAATATTGT(SEQ ID NO.24);

[0109] TCGTTAAATAGATACGTTACGC (SEQ ID NO. 33);

[0110] TAAAAACTAAAAACTTTCCCGG (SEQ ID NO. 34);

[0111] TCGTTAAATAGATACGTTACGC (SEQ ID NO. 35);

[0112] CAACGCCTCGAAACCTACG (SEQ ID NO. 36).

[0113] CCCCCAGGATTCTTACAGAAAACAAGTGGT (SEQ ID NO. 38);

[0114] GCAAATACACAGAGGAAGCCTTCGCCTGTCCTC (SEQ ID NO. 39);

[0115] CAAGTGGTTATAGATGGTGA (SEQ ID NO. 40);

[0116] CGCCTGTCCTCATGTATTGG (SEQ ID NO. 41).

[0117] More preferably, the enzyme comprises: a DNA polymerase and / or a recombinase.

[0118] More preferably, the auxochromic group comprises MgOAc.

[0119] Preferably, the above reaction system can be used to enrich mutant alleles with MAF ≥ 0.01%.

[0120] The present invention also discloses the use of the reaction system, including at least one of the following:

[0121] (1) Targeted binding to target nucleic acid fragments;

[0122] (2) Targeted cleavage of target nucleic acid fragments;

[0123] (3) Enrichment of mutant genes;

[0124] (4) Detection of mutant genes;

[0125] (5) Enrichment of methylated DNA; or,

[0126] (6) Detection of methylated DNA.

[0127] Preferably, the above-mentioned enrichment includes automated enrichment; and the above-mentioned detection includes automated detection.

[0128] More preferably, the automated enrichment comprises enrichment using a microfluidic chip; and the automated detection comprises detection using a microfluidic chip.

[0129] More preferably, the microfluidic chip is divided into three parts, consisting of a top packaging sheet, a bottom packaging sheet and a middle reaction layer.

[0130] More preferably, the top packaging sheet structure includes: a microfluidic chip mounting hole; a microfluidic chip packaging positioning hole; and a microfluidic chip sampling hole.

[0131] More preferably, the intermediate reaction layer structure includes: microfluidic chip packaging positioning hole; microfluidic chip mounting hole; pre-amplification reaction chamber; siphon valve; digestion reaction chamber; pre-distribution chamber; PCR reaction chamber; waste liquid chamber; capillary valve; 10: gas passage.

[0132] More preferably, the bottom packaging sheet structure includes: a microfluidic chip mounting hole; a microfluidic chip packaging positioning hole; an RNaseA sample adding cavity; and a ProteinaseK sample adding cavity.

[0133] Preferably, the mutated gene includes a cancer-related gene.

[0134] Preferably, the cancer-related genes include at least one of the following:

[0135] EGFR, BRAF, PIK3CA, TP53, LRP1B, APC, CYP1A1, NP01, EPHX1, KRAS, BRCA1, BRCA2, MET, MLH1, MSH2, MSH3, MSH6, PALB2, BMPR1A, SMAD4, STK11, PTEN, AXIN 2, BLM, BUB1B, CDH1, CEP57, CHEK2, ENG, EPCAM, FLCN, GALNTI2, GREM1, FAT4, KMT2D, KMT2C, ARID1A, FAT1, PTEN, ATM, ZFHX3, CREBBP, GRIN2A, NRAS or NF1.

[0136] Preferably, the methylated DNA includes cancer-associated methylated genes and / or their promoters.

[0137] Preferably, the cancer-related methylation genes include at least one of the following:

[0138] PCDH-10, BRCA1, RASSF1A, ESR1, APC, p14ARF, p16INK4a, DAPK, CDH1, RUNX3, TFPI2, SFRP5, HI C1, PAX5, PGR, THBS1, ESR, COL23A1, C2CD4D, WNT6, OPCML, ZNF154, RARb2, ATM, MGMT, GSTP1, MI R129-2, LINC01158, CCDC181, PRKCB, TBR1, ZNF781, MARCH11, VWC2, SLC9A3, HOXA7, Septin9, I KZF1, BCAT1, hMLH1, WIF1, CDKN2A, SHOX2, 3OST2, ASSF1A, RARb, PITX2, NID2, NEUROG2 or HOXA1;

[0139] The promoter includes at least one of the promoters of the following genes:

[0140] PCDH-10, BRCA1, RASSF1A, ESR1, APC, p14ARF, p16INK4a, DAPK, CDH1, RUNX3, TFPI2, SFRP5, HI C1, PAX5, PGR, THBS1, ESR, COL23A1, C2CD4D, WNT6, OPCML, ZNF154, RARb2, ATM, MGMT, GSTP1, MI R129-2, LINC01158, CCDC181, PRKCB, TBR1, ZNF781, MARCH11, VWC2, SLC9A3, HOXA7, Septin9, I KZF1, BCAT1, hMLH1, WIF1, CDKN2A, SHOX2, 3OST2, ASSF1A, RARb, PITX2, NID2, NEUROG2 or HOXA1.

[0141] The present invention also discloses the use of the ribonucleoprotein complex, including at least one of the following:

[0142] (1) Enrichment of gene mutations;

[0143] (2) Detection of gene mutations;

[0144] (3) Enrichment of methylated DNA; or,

[0145] (4) Detection of methylated DNA.

[0146] Preferably, the genes include: EGFR, BRAF, PIK3CA, TP53, LRP1B, APC, CYP1A1, NP01, EPHX1, KRAS, BRCA1, BRCA2, MET, MLH1, MSH2, MSH3, MSH6, PALB2, BMPR1A, SMAD4, STK11, PTEN, AXIN2, BLM, BUB1B, CDH1, CEP57, CHEK2, ENG, EPCAM, FLCN, GALNTI2, GREM1, FAT4, KMT2D, KMT2C, ARID1A, FAT1, PTEN, ATM, ZFHX3, CREBBP, GRIN2A, NRAS or NF1.

[0147] The present invention also discloses uses of Cas proteins, including at least one of the following:

[0148] (1) Enrichment of gene mutations;

[0149] (2) Detection of gene mutations;

[0150] (3) Enrichment of methylated DNA; or,

[0151] (4) Detection of methylated DNA.

[0152] The present invention also discloses the use of Ago protein, including at least one of the following:

[0153] (1) Enrichment of gene mutations;

[0154] (2) Detection of gene mutations;

[0155] (3) Enrichment of methylated DNA; or,

[0156] (4) Detection of methylated DNA.

[0157] Preferably, the genes include at least one of the following: EGFR, BRAF, PIK3CA, TP53, LRP1B, APC, CYP1A1, NP01, EPHX1, KRAS, BRCA1, BRCA2, MET, MLH1, MSH2, MSH3, MSH6, PALB2, BMPR1A, SMAD4, STK11, PTEN, AXIN2, BLM, BUB1B, CDH1, CEP57, CHEK2, ENG, EPCAM, FLCN, GALNTI2, GREM1, FAT4, KMT2D, KMT2C, ARID1A, FAT1, PTEN, ATM, ZFHX3, CREBBP, GRIN2A, NRAS or NF1.

[0158] Preferably, the Cas protein includes at least one of the following: Cas12a, SacCas9, CjCas9, SpCas9, NmCas9, Sp-Cas9 HF1, evoCas9, HypaCas9, HiFi Cas9, Sniper-Cas9, xCas9, eSpCas9 1.1, SuperFiCas9, SaCas9, SaCas9-HF, efSaCas9, ScCas9, Cas9—Sc++, Cas9 HiFi-Sc++, SpaCas9, SpaCas9-HF, FnCas9, AnaCas9, SpyCas9, FnCas12a, LbCas12a or AsCas12a.

[0159] Preferably, the Ago protein comprises at least one of the following: CbAgo, TtAgo, PfAgo, KmAgo or KpAgo.

[0160] The present invention also discloses a kit for enriching mutant genes of programmable enzymes, and the kit comprises the above-mentioned ribonucleoprotein complex.

[0161] Preferably, the ribonucleoprotein complex comprises:

[0162] Component (a), comprising a nucleic acid region capable of complementary pairing with a target nucleic acid and a 0-4 bp mismatch fragment;

[0163] Component (b), capable of binding to the target nucleic acid and breaking the target nucleic acid strand;

[0164] Wherein, component (a) is selected from tracrRNA and / or crRNA and / or sgRNA and / or tracrRNA derivatives and / or crRNA derivatives and / or sgRNA derivatives and / or guide DNA and / or guide RNA; component (b) includes Cas protein and / or Cas protein derivatives and / or Ago protein and / or Ago protein derivatives; components (a) and (b) can be combined.

[0165] Preferably, the length of the mismatched nucleotide fragment is 0 bp, 1 bp, 2 bp, 3 bp or 4 bp.

[0166] Preferably, component (a) carries a label, the label comprising streptavidin or biotin.

[0167] More preferably, component (a) carries a chemical modification comprising a sulfur-substituted oxygen or a methoxy modification.

[0168] More preferably, the sgRNA in component (a) includes a mismatched nucleotide fragment.

[0169] More preferably, the spacer sequence length of the sgRNA in component (a) is 16-22 nt.

[0170] More preferably, the sgRNA sequence is as follows:

[0171] UCUUAAUUCCUUGAUAGCGA (SEQ ID NO.1);

[0172] UAGCUACAGUGAACUCUCGA (SEQ ID NO.2);

[0173] UAGCUACAGUGAAAUCACGA (SEQ ID NO.3);

[0174] GCUACAGUGAACUCUCGA (SEQ ID NO.4);

[0175] GUCUAGCUACAGUGAAA (SEQ ID NO. 5);

[0176] GUCUAGCUGCAGUGAAA (SEQ ID NO. 6);

[0177] GGCAGCCGAAGGGCAUGAGC (SEQ ID NO. 27);

[0178] GGCAGCCGAAGAGCAUGAGC (SEQ ID NO. 28);

[0179] AAUUUUUGUUUGAGUGGUUG (SEQ ID NO. 37);

[0180] UCCAGCUGUAUCCAGUAUGU (SEQ ID NO. 42).

[0181] More preferably, the guide DNA sequence is as follows:

[0182] Forward guide: p-TAGATTTCACTGTAGC-3' (SEQ ID NO. 43);

[0183] Reverse guide: p-TTCTAGCTACAGTGAA-3' (SEQ ID NO. 44).

[0184] More preferably, component (b) includes at least one of the following: Cas12a, SacCas9, CjCas9, SpCas9, NmCas9, Sp-Cas9 HF1, evoCas9, HypaCas9, HiFi Cas9, Sniper-Cas9, xCas9, eSpCas9 1.1, SuperFiCas9, SaCas9, SaCas9-HF, efSaCas9, ScCas9, Cas9—Sc++, Cas9 HiFi-Sc++, SpaCas9, SpaCas9-HF, FnCas9, AnaCas9, SpyCas9, FnCas12a, LbCas12a, AsCas12a, CbAgo, TtAgo, PfAgo, KmAgo or KpAgo.

[0185] More preferably, Cas12a is from at least one of the following strains: Francisella novicida ; Acidaminococcus ; Lachnospiraceae .

[0186] More preferably, SacCas9 is from a bacterial species Staphylococcus aureus .

[0187] More preferably, CjCas9 is from a bacterial species Campylobacter jejuni .

[0188] More preferably, SpCas9 is derived from a bacterial species Streptococcus pyogenes .

[0189] More preferably, NmCas9 is from a bacterial species Neisseria meningitidis .

[0190] More preferably, SaCas9 is derived from a bacterial species Staphylococcus aureus.

[0191] More preferably, ScCas9 is derived from a bacterial species Streptococcus canis.

[0192] More preferably, SpaCas9 is derived from a bacterial species Streptococcus pasteurianus.

[0193] More preferably, in the ribonucleoprotein complex, the final concentration of component (a) is 0.1-2 μM.

[0194] More preferably, in the ribonucleoprotein complex, the final concentration of component (b) is 0.1-3 μM.

[0195] More preferably, the ribonucleoprotein complex further comprises a hydrogen ion buffer.

[0196] More preferably, in the ribonucleoprotein complex, the final concentration of the hydrogen ion buffer is 1 mM-2 M.

[0197] Preferably, the kit also includes a constant temperature cutting-amplification reaction system.

[0198] Preferably, in the reaction system, the final concentration of the ribonucleoprotein complex is 0.1-2 μM.

[0199] Preferably, the reaction system further comprises: primers, target gene, enzyme and auxochromogen.

[0200] Preferably, the reaction system further comprises: dNTP and single-stranded binding protein.

[0201] More preferably, the target gene includes: genomic DNA or cell-free DNA.

[0202] More preferably, the target gene comprises genomic DNA extracted from at least one of the following cell lines:

[0203] EGFR 19del wild-type cell line; EGFR 19 E746_A750 del (2235-2249del) cell line; EGFR19 E746_A750 del (2236-2250del); BRAF V600E mutant; BRAFV600E wild-type cell line; B-CPAP cell line.

[0204] More preferably, the primer comprises a nucleotide sequence capable of binding to the gene of interest and directing synthesis.

[0205] More preferably, the primer comprises at least one of the following sequences:

[0206] GCATGTGGCACCATCTCACA (SEQ ID NO. 15);

[0207] AGAGCAGCTGCCAGACATGA (SEQ ID NO. 16);

[0208] CTACACCTCAGATATATTTC(SEQ ID NO.19);

[0209] TGGATCCAGACAACTGT(SEQ ID NO.20);

[0210] TACGTGATGGCCAGCGTGGA (SEQ ID NO. 23);

[0211] ACTGGGAGCCAATATTGT(SEQ ID NO.24);

[0212] TCGTTAAATAGATACGTTACGC (SEQ ID NO. 33);

[0213] TAAAAACTAAAAACTTTCCCGG (SEQ ID NO. 34);

[0214] TCGTTAAATAGATACGTTACGC (SEQ ID NO. 35);

[0215] CAACGCCTCGAAACCTACG (SEQ ID NO. 36).

[0216] CCCCCAGGATTCTTACAGAAAACAAGTGGT (SEQ ID NO. 38);

[0217] GCAAATACACAGAGGAAGCCTTCGCCTGTCCTC (SEQ ID NO. 39);

[0218] CAAGTGGTTATAGATGGTGA (SEQ ID NO. 40); or,

[0219] CGCCTGTCCTCATGTATTGG (SEQ ID NO. 41).

[0220] More preferably, the enzyme comprises: a DNA polymerase and / or a recombinase.

[0221] More preferably, the auxochromic group comprises MgOAc.

[0222] Preferably, the above reaction system can be used to enrich mutant alleles with MAF ≥ 0.01%.

[0223] The present invention also discloses a method for detecting and / or enriching low-abundance mutant genes using the above kit, the operating steps comprising:

[0224] Collect samples;

[0225] preparing ribonucleoprotein complexes;

[0226] Prepare the reaction system;

[0227] Enrichment analysis.

[0228] Preferably, the sample collecting step comprises extracting DNA and / or RNA from the sample using a nucleic acid extraction kit.

[0229] More preferably, the sample comprises at least one of the following: blood, plasma / serum, cerebrospinal fluid, urine, saliva.

[0230] More preferably, the sample comprises at least one of the following obtained from a cancer patient: blood, plasma / serum, cerebrospinal fluid, urine, saliva.

[0231] Preferably, the step of preparing the ribonucleoprotein complex comprises:

[0232] Cas9, sgRNA and hydrogen ion buffer were mixed and incubated.

[0233] Preferably, the hydrogen ion buffer comprises HEPES.

[0234] Preferably, in the preparation of the ribonucleoprotein complex, the final concentration of Cas9 used is 0.1-3 μM.

[0235] More preferably, in the preparation of the ribonucleoprotein complex, the final concentration of Cas9 used is 2.5 μM.

[0236] Preferably, in the preparation of the ribonucleoprotein complex, the final concentration of the sgRNA used is 0.1-2 μM.

[0237] More preferably, in the preparation of the ribonucleoprotein complex, the final concentration of the sgRNA used is 2.5 μM.

[0238] Preferably, in the preparation of the ribonucleoprotein complex, the final concentration of HEPES used is 1 mM-2M.

[0239] More preferably, in the preparation of the ribonucleoprotein complex, the final concentration of HEPES used is 1M.

[0240] Preferably, in the preparation of the ribonucleoprotein complex, the reaction temperature is 34-39° C. and the reaction time is 3-18 min.

[0241] More preferably, in the preparation of the ribonucleoprotein complex, the reaction temperature is 37° C. and the reaction time is 10 min.

[0242] Preferably, the step of preparing a reaction system includes preparing a constant temperature cleavage-amplification reaction system.

[0243] More preferably, in the reaction system, the final concentration of the ribonucleoprotein complex is 0.1-2 μM.

[0244] More preferably, the reaction system further comprises: primers, target genes, enzymes and auxochromic groups.

[0245] More preferably, the reaction system further comprises: dNTP and single-stranded binding protein.

[0246] More preferably, the target gene includes: genomic DNA.

[0247] More preferably, the target gene comprises genomic DNA extracted from at least one of the following cell lines:

[0248] EGFR 19del wild-type cell line; EGFR 19 E746_A750 del (2235-2249del) cell line; EGFR19 E746_A750 del (2236-2250del); BRAF V600E mutant; BRAFV600E wild-type cell line; B-CPAP cell line.

[0249] More preferably, the primer comprises a nucleotide sequence capable of binding to the target gene and guiding synthesis.

[0250] More preferably, the primer comprises at least one of the following sequences:

[0251] GCATGTGGCACCATCTCACA (SEQ ID NO. 15);

[0252] AGAGCAGCTGCCAGACATGA (SEQ ID NO. 16);

[0253] CTACACCTCAGATATATTTC(SEQ ID NO.19);

[0254] TGGATCCAGACAACTGT(SEQ ID NO.20);

[0255] TACGTGATGGCCAGCGTGGA (SEQ ID NO. 23);

[0256] ACTGGGAGCCAATATTGT(SEQ ID NO.24);

[0257] TCGTTAAATAGATACGTTACGC (SEQ ID NO. 33);

[0258] TAAAAACTAAAAACTTTCCCGG (SEQ ID NO. 34);

[0259] TCGTTAAATAGATACGTTACGC (SEQ ID NO. 35);

[0260] CAACGCCTCGAAACCTACG (SEQ ID NO. 36).

[0261] CCCCCAGGATTCTTACAGAAAACAAGTGGT (SEQ ID NO. 38);

[0262] GCAAATACACAGAGGAAGCCTTCGCCTGTCCTC (SEQ ID NO. 39);

[0263] CAAGTGGTTATAGATGGTGA (SEQ ID NO. 40); or,

[0264] CGCCTGTCCTCATGTATTGG (SEQ ID NO. 41).

[0265] More preferably, the enzyme comprises: DNA polymerase and / or recombinase.

[0266] Still more preferably, the auxochromic group comprises MgOAc.

[0267] Preferably, the enrichment analysis step includes QPCR analysis, sequencing, point-of-care testing (POCT), and the above-mentioned programmable nuclease-based detection method for low-abundance mutant genes.

[0268] More preferably, the QPCR analysis step comprises preparing a QPCR reaction system.

[0269] More preferably, the QPCR reaction system comprises:

[0270] Primers, auxochromogens, dNTPs, enzymes.

[0271] More preferably, the primer comprises at least one of the following:

[0272] TGTCATAGGGACTCTGGATCCCAGA (SEQ ID NO. 17);

[0273] GCAGAAACTCACATCGAGGATTTCCTGTT (SEQ ID NO. 18);

[0274] CCTCAGATATATTTCTTCATGA (SEQ ID NO. 21);

[0275] TGTTCAAACTGATGGGAC (SEQ ID NO. 22);

[0276] TGATGGCCAGCGTGGACAA (SEQ ID NO. 25); or

[0277] TTGTGTTCCCGGACATAGTC (SEQ ID NO. 26).

[0278] Still more preferably, the auxochromic group comprises MgOAc.

[0279] Even more preferably, the enzyme comprises a DNA polymerase.

[0280] The present invention also discloses a design method of sgRNA based on the CRISPR system, wherein the sgRNA comprises a specific recognition nucleotide fragment and a mismatched nucleotide fragment; wherein the length of the mismatched nucleotide fragment is 0-4 bp.

[0281] Preferably, the specific recognition nucleotide fragment is a spacer sequence.

[0282] More preferably, the spacer sequence length is 18-22 nt.

[0283] Preferably, the length of the mismatched nucleotide fragment is 0 bp, 1 bp, 2 bp, 3 bp or 4 bp.

[0284] The sgRNA designed using the sgRNA design method of the present invention can effectively distinguish wild-type alleles from mutant alleles., and the sgRNA designed using the sgRNA design method of the present invention has high spacer sequence recognition and cutting efficiency. The mismatched nucleotide fragments designed by the present invention are conducive to expanding the scope of use of sgRNA, and can enable sgRNA to recognize more mutations.

[0285] The present invention also discloses an sgRNA, which comprises a specific recognition nucleotide fragment and a mismatched nucleotide fragment; wherein the mismatched nucleotide fragment has a length of 0-4 bp.

[0286] Preferably, the length of the mismatched nucleotide fragment is 0 bp, 1 bp, 2 bp, 3 bp or 4 bp.

[0287] Preferably, the above sgRNA is designed using the above method.

[0288] Preferably, the site of the mismatched nucleotide is in the first 5 bp of the mutant gene and / or the last 5 bp of the mutant gene.

[0289] Preferably, the specific recognition nucleotide fragment is a spacer sequence.

[0290] More preferably, the spacer sequence length is 18-22 nt.

[0291] More preferably, the sgRNA sequence is as follows:

[0292] UCUUAAUUCCUUGAUAGCGA (SEQ ID NO.1);

[0293] UAGCUACAGUGAACUCUCGA (SEQ ID NO.2);

[0294] UAGCUACAGUGAAAUCACGA (SEQ ID NO.3);

[0295] GCUACAGUGAACUCUCGA (SEQ ID NO.4);

[0296] GUCUAGCUACAGUGAAA (SEQ ID NO. 5);

[0297] GUCUAGCUGCAGUGAAA (SEQ ID NO. 6);

[0298] GGCAGCCGAAGGGCAUGAGC (SEQ ID NO. 27);

[0299] GGCAGCCGAAGAGCAUGAGC (SEQ ID NO. 28);

[0300] AAUUUUUGUUUGAGUGGUUG (SEQ ID NO. 37);

[0301] UCCAGCUGUAUCCAGUAUGU (SEQ ID NO. 42).

[0302] The present invention also discloses the use of the above sgRNA, including at least one of the following:

[0303] (1) Enrichment of gene mutations;

[0304] (2) Detection of gene mutations;

[0305] (3) Enrichment of methylated DNA; or,

[0306] (4) Detection of methylated DNA.

[0307] The present invention also discloses a guide DNA or guide RNA, characterized in that the guide DNA or guide RNA comprises a specific recognition nucleotide fragment and a mismatched nucleotide fragment; wherein the length of the mismatched nucleotide fragment is 0-4bp.

[0308] Preferably, the length of the mismatched nucleotide fragment is 0 bp, 1 bp, 2 bp, 3 bp or 4 bp.

[0309] Preferably, the guide DNA comprises the following sequence:

[0310] SEQ ID NO.43: Forward guide: p-TAGATTTCACTGTAGC-3';

[0311] SEQ ID NO.44: Reverse guide: p-TTCTAGCTACAGTGAA-3'.

[0312] The present invention also discloses the use of the guide DNA or guide RNA, including at least one of the following:

[0313] (1) Enrichment of gene mutations;

[0314] (2) Detection of gene mutations;

[0315] (3) Enrichment of methylated DNA; or,

[0316] (4) Detection of methylated DNA.

[0317] The present invention also discloses a method for enriching mutant genes and / or methylated DNA based on a programmable enzyme, using a CRISPR system to specifically cut wild-type alleles and simultaneously amplify mutant alleles; wherein the CRISPR system comprises:

[0318] Component (a), comprising a nucleic acid region capable of complementary pairing with a target nucleic acid and a 0-4 bp mismatch fragment;

[0319] Component (b), capable of binding to the target nucleic acid and breaking the target nucleic acid strand;

[0320] Wherein, component (a) is selected from tracrRNA and / or crRNA and / or sgRNA and / or tracrRNA derivatives and / or crRNA derivatives and / or sgRNA derivatives and / or guide DNA and / or guide RNA; component (b) includes Cas protein and / or Cas protein derivatives and / or Ago protein and / or Ago protein derivatives; components (a) and (b) can be combined. Preferably, component (a) carries a label, and the label includes streptavidin or biotin.

[0321] Preferably, the length of the mismatched nucleotide fragment is 0 bp, 1 bp, 2 bp, 3 bp or 4 bp.

[0322] Preferably, component (a) carries a chemical modification comprising a sulfur-substituted oxygen or a methoxy modification.

[0323] Preferably, the sgRNA in component (a) includes a mismatched nucleotide fragment. The mismatched nucleotide fragment designed in the present invention is conducive to expanding the scope of use of sgRNA and enables sgRNA to recognize more mutations.

[0324] Preferably, the spacer sequence length of the sgRNA in component (a) is 16-22 nt.

[0325] Preferably, component (b) includes at least one of the following: Cas12a, SacCas9, CjCas9, SpCas9, NmCas9, Sp-Cas9 HF1, evoCas9, HypaCas9, HiFi Cas9, Sniper-Cas9, xCas9, eSpCas9 1.1, SuperFiCas9, SaCas9, SaCas9-HF, efSaCas9, ScCas9, Cas9-Sc++, Cas9 HiFi-Sc++, SpaCas9, SpaCas9-HF, FnCas9, AnaCas9, SpyCas9, FnCas12a, LbCas12a, AsCas12a, CbAgo, TtAgo, PfAgo, KmAgo or KpAgo.

[0326] More preferably, Cas12a is from at least one of the following species: Francisella novicida ; Acidaminococcus ; Lachnospiraceae .

[0327] More preferably, SacCas9 is from a bacterial species Staphylococcus aureus .

[0328] More preferably, CjCas9 is from a bacterial species Campylobacter jejuni .

[0329] More preferably, SpCas9 is derived from a bacterial species Streptococcus pyogenes .

[0330] More preferably, NmCas9 is derived from a bacterial species Neisseria meningitidis .

[0331] More preferably, SaCas9 is derived from a bacterial species Staphylococcus aureus.

[0332] More preferably, ScCas9 is derived from a bacterial species Streptococcus canis.

[0333] More preferably, SpaCas9 is derived from a bacterial species Streptococcus pasteurianus.

[0334] Preferably, in the CRISPR system, the final concentration of component (a) is 0.1-2 μM.

[0335] Preferably, in the CRISPR system, the final concentration of component (b) is 0.1-3 μM.

[0336] Preferably, the CRISPR system also includes a hydrogen ion buffer.

[0337] More preferably, in the CRISPR system, the hydrogen ion buffer comprises: HEPES.

[0338] More preferably, in the CRISPR system, the final concentration of the hydrogen ion buffer is 1 mM-2 M.

[0339] Preferably, the method for amplifying the mutant allele includes PCR, LAMP, and RPA.

[0340] More preferably, the reaction system for amplifying the mutant allele comprises a ribonucleoprotein complex.

[0341] Preferably, in the reaction system, the final concentration of the ribonucleoprotein complex is 0.1-2 μM.

[0342] Preferably, the reaction system further comprises: primers, target gene, enzyme and auxochromogen.

[0343] Preferably, the reaction system further comprises: dNTP and single-stranded binding protein.

[0344] More preferably, the target gene includes: genomic DNA.

[0345] More preferably, the target gene comprises genomic DNA extracted from at least one of the following cell lines:

[0346] EGFR 19del wild-type cell line; EGFR 19 E746_A750 del (2235-2249del) cell line; EGFR19 E746_A750 del (2236-2250del); BRAF V600E mutant; BRAFV600E wild-type cell line; B-CPAP cell line.

[0347] More preferably, the primer comprises a nucleotide sequence capable of binding to the gene of interest and directing synthesis.

[0348] More preferably, the primer comprises at least one of the following sequences:

[0349] GCATGTGGCACCATCTCACA (SEQ ID NO. 15);

[0350] AGAGCAGCTGCCAGACATGA (SEQ ID NO. 16);

[0351] CTACACCTCAGATATATTTC(SEQ ID NO.19);

[0352] TGGATCCAGACAACTGT(SEQ ID NO.20);

[0353] TACGTGATGGCCAGCGTGGA (SEQ ID NO. 23);

[0354] ACTGGGAGCCAATATTGT(SEQ ID NO.24);

[0355] TCGTTAAATAGATACGTTACGC (SEQ ID NO. 33);

[0356] TAAAAACTAAAAACTTTCCCGG (SEQ ID NO. 34);

[0357] TCGTTAAATAGATACGTTACGC (SEQ ID NO. 35);

[0358] CAACGCCTCGAAACCTACG (SEQ ID NO. 36).

[0359] CCCCCAGGATTCTTACAGAAAACAAGTGGT (SEQ ID NO. 38);

[0360] GCAAATACACAGAGGAAGCCTTCGCCTGTCCTC (SEQ ID NO. 39);

[0361] CAAGTGGTTATAGATGGTGA (SEQ ID NO. 40); or,

[0362] CGCCTGTCCTCATGTATTGG (SEQ ID NO. 41).

[0363] More preferably, the guide DNA sequence is as follows:

[0364] Forward guide: p-TAGATTTCACTGTAGC-3' (SEQ ID NO. 43);

[0365] Reverse guide: p-TTCTAGCTACAGTGAA-3' (SEQ ID NO. 44).

[0366] More preferably, the enzyme comprises: a DNA polymerase and / or a recombinase.

[0367] More preferably, the auxochromic group comprises MgOAc.

[0368] Still more preferably, the ribonucleoprotein complex comprises Cas9, sgRNA and a hydrogen ion buffer.

[0369] More preferably, the method for preparing the ribonucleoprotein complex comprises: mixing and incubating Cas9, sgRNA and a hydrogen ion buffer.

[0370] Still more preferably, the hydrogen ion buffer comprises HEPES.

[0371] More preferably, in the preparation of the ribonucleoprotein complex, the final concentration of Cas9 used is 0.1-3 μM.

[0372] More preferably, in the preparation of the ribonucleoprotein complex, the final concentration of the sgRNA used is 0.1-2 μM.

[0373] More preferably, in the preparation of the ribonucleoprotein complex, the final concentration of HEPES used is 1 mM-2M.

[0374] More preferably, in the preparation of the ribonucleoprotein complex, the reaction temperature is 34-39° C. and the reaction time is 3-18 min.

[0375] The present invention also discloses a method for detecting low-abundance mutant genes and / or methylated DNA based on programmable nucleases, using the above-mentioned CRISPR system or Ago system to specifically cut the wild-type allele and simultaneously amplify the mutant allele; comprising the following steps:

[0376] Design sgRNA;

[0377] Acquisition of target genes;

[0378] Amplification and cleavage;

[0379] Mutation gene testing.

[0380] Preferably, the sgRNA includes mismatched nucleotide fragments.

[0381] Preferably, the spacer sequence length of sgRNA is 16-22 nt.

[0382] Preferably, the target gene includes EGFR, BRAF, PIK3CA, TP53, LRP1B, APC, CYP1A1, NP01, EPHX1, KRAS, BRCA1, BRCA2, MET, MLH1, MSH2, MSH3, MSH6, PALB2, BMPR1A, SMAD4, STK11, PTEN, AXIN2, BLM, BUB1B, CDH1, CEP57, CHEK2, ENG, EPCAM, FLCN, GALNTI2, GREM1, FAT4, KMT2D, KMT2C, ARID1A, FAT1, PTEN, ATM, ZFHX3, CREBBP, GRIN2A, NRAS or NF1.

[0383] Preferably, the amplification method includes: PCR, LAMP and RPA.

[0384] Preferably, the mutant gene detection method includes Sanger sequencing.

[0385] Preferably, the reaction system used for amplification includes: target gene, ribonucleoprotein complex, MgOAc, primers, DNA polymerase, recombinase and single-stranded binding protein.

[0386] Preferably, the reaction system used for cleavage includes: target gene, ribonucleoprotein complex, and MgOAc.

[0387] The invention also discloses a microfluidic chip for automatically enriching and / or detecting mutant genes and / or methylated DNA.

[0388] Preferably, the microfluidic chip is divided into three parts, consisting of a top packaging sheet, a bottom packaging sheet and a middle reaction layer.

[0389] More preferably, the top packaging sheet structure includes: a microfluidic chip mounting hole; a microfluidic chip packaging positioning hole; and a microfluidic chip sampling hole.

[0390] More preferably, the intermediate reaction layer structure includes: microfluidic chip packaging positioning hole; microfluidic chip mounting hole; pre-amplification reaction chamber; siphon valve; digestion reaction chamber; pre-distribution chamber; PCR reaction chamber; waste liquid chamber; capillary valve; 10: gas passage.

[0391] More preferably, the bottom packaging sheet structure includes: a microfluidic chip mounting hole; a microfluidic chip packaging positioning hole; an RNaseA sample addition cavity; and a ProteinaseK sample addition cavity.

[0392] The present invention also discloses a method for automatically enriching and / or detecting mutant genes and / or methylated DNA, comprising the following steps:

[0393] 1) Sample addition: add the pre-amplification system to the pre-amplification reaction chamber; add RNaseA to the RNaseA sample chamber; add ProteinaseK to the ProteinaseK sample chamber; add the qPCR system to the PCR reaction chamber;

[0394] 2) Sealing;

[0395] 3) Load and complete the experiment: Load the sealed microfluidic chip onto the centrifugal microfluidic platform.

[0396] Compared with the prior art, the present invention has the following beneficial effects:

[0397] The present invention is based on the CRISPR system, programmable nuclease and nucleic acid amplification technology. While amplifying nucleic acids, programmable nucleases are used to specifically cut wild-type nucleic acids, so that the number of mutant alleles in the sample increases exponentially continuously, reaching a level that can be detected by cheap Sanger sequencing. Not only has it achieved a detection sensitivity comparable to ultra-deep NGS sequencing, but it also has the characteristics of a short detection cycle and no need to rely on large-scale instruments and equipment, which greatly reduces the detection cost and greatly promotes the clinical application of low-abundance mutant allele detection. The enrichment method of the present invention can be used to enrich mutant alleles with MAF ≥ 0.01%, and can significantly increase the frequency of mutant alleles. BRIEF DESCRIPTION OF THE DRAWINGS

[0398] Figure 1 The schematic diagram of the present invention for enriching low-abundance mutant alleles;

[0399] Figure 2 This is a diagram of the sgRNA cutting verification results in Example 1;

[0400] Figure 3 The sequencing results of the wild-type allele and ED2 before and after ED2 was treated with SpCas9 in Example 1;

[0401] Figure 4The sequencing result of ED2 after ED2 was treated with Hypa Cas9 in Example 1;

[0402] Figure 5 The sequencing results of the wild-type allele and ED1 before and after ED1 was treated with SpCas9 HF1 in Example 1;

[0403] Figure 6 The sequencing result of ED1 after ED1 was treated with Evo Cas9 in Example 1;

[0404] Figure 7 This is a diagram of the sgRNA cutting verification results in Example 2;

[0405] Figure 8 The sequencing results of wild-type alleles and mutant alleles before and after HiFi Cas9 treatment in Example 2;

[0406] Fig. 9 This is the sequencing result of the mutant allele after treatment with SuperFi Cas9 in Example 2;

[0407] Fig.10 This is the result of 0min mutant allele sequencing using HiFi SC++ in Example 2;

[0408] Fig.11 This is the mutant allele sequencing result using HiFi SC++ for 5 minutes in Example 2;

[0409] Fig.12 This is the mutant allele sequencing result using HiFi SC++ for 10 minutes in Example 2;

[0410] Fig.13 This is the mutant allele sequencing result using HiFi SC++ for 15 minutes in Example 2;

[0411] Fig.14 This is the mutant allele sequencing result using HiFi SC++ for 20 minutes in Example 2;

[0412] Fig.15 This is a diagram of the sgRNA cutting verification results in Example 3;

[0413] Fig.16 The sequencing results of the wild-type allele and mutant allele before and after treatment with Cas9 in Example 3;

[0414] Fig.17 Comparison of the enrichment degree of 0.1% methylated DNA before and after enrichment treatment in Example 4;

[0415] Fig.18 It is a schematic diagram of the principle of enriching methylated DNA in the present invention;

[0416] Fig.19 The sequencing results of wild-type alleles and mutant alleles before and after NRAS treatment using Cas12 in Example 5;

[0417] Fig. 20 Schematic diagram of the microfluidic chip structure;

[0418] Fig.21 It is a schematic diagram of the top package structure;

[0419] Fig. 22 Schematic diagram of the structure of the intermediate reaction layer;

[0420] Fig.23 It is a schematic diagram of the top package structure;

[0421] Fig.24 This is a schematic diagram of the present invention based on Ago cutting;

[0422] Fig.25 This is a comparison of MAF=0.1% in Example 7 before and after PASEA treatment.

[0423] Figure numbers: 1-top package sheet;

[0424] 11-microfluidic chip mounting hole; 12-microfluidic chip packaging positioning hole; 13-microfluidic chip injection hole;

[0425] 2-intermediate reaction layer;

[0426] 21-microfluidic chip packaging positioning hole; 22-microfluidic chip mounting hole; 23-preamplification reaction chamber; 24-siphon valve; 25-digestion reaction chamber; 26-predistribution chamber; 27-PCR reaction chamber; 28-waste liquid chamber; 29-capillary valve; 210-gas passage;

[0427] 3- bottom packaging sheet;

[0428] 31- microfluidic chip installation hole; 32- microfluidic chip packaging positioning hole; 33- RNaseA sample addition chamber; 34- ProteinaseK sample addition chamber. DETAILED DESCRIPTION

[0429] Here, exemplary embodiments will be described in detail, and the embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present disclosure. Instead, they are only examples of methods consistent with some aspects of the present disclosure.

[0430] The experimental methods in the following examples, unless otherwise specified, are all conventional methods or conditions recommended by the manufacturer. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0431] The primers used for sequencing in the following examples are the same as the qPCR primers.

[0432] Example 1

[0433] Enrichment of EGFR gene del19 mutation

[0434] 1. Primer design

[0435] The gene sequence of exon 19 of human EGFR gene was obtained from NCBI database, and primers were designed based on the sequence:

[0436] 19del preamplification F primer: GCATGTGGCACCATCTCACA (SEQ ID NO. 15);

[0437] 19del preamplification R primer: AGAGCAGCTGCCAGACATGA (SEQ ID NO. 16);

[0438] 19del qPCR F primer: TGTCATAGGGACTCTGGATCCCAGA (SEQ ID NO.17);

[0439] 19del qPCR R primer: GCAGAAACTCACATCGAGGATTTCCTTGT (SEQ ID NO. 18).

[0440] 2. Design sgRNA

[0441] Design an sgRNA sequence with a length of 20 nt that can completely match the wild-type allele sequence, as shown in SEQ ID NO.1:

[0442] UCUUAAUUCCUUGAUAGCGA (SEQ ID NO. 1).

[0443] Use sgRNA to guide Cas protein to bind to the wild-type allele and specifically cut the wild-type allele without cutting the mutant allele. The principle is as follows Figure 1 shown.

[0444] 3. Acquisition of target genes

[0445] The following is the short gene sequence used as a marker in this example:

[0446] EGFR-WT-S:

[0447] CTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAATTAAGAGAAGCAACATCTCCGAAAGCCAACAAGGAAATCCTCGATGTGA(SEQ ID NO.7);

[0448] EGFR-WT-AS:

[0449] TCACATCGAGGATTTCCTTGTTGGCTTTCGGAGATGTTGCTTCTCTTAATTCCTTGATAGCGACGGGAATTTTAACTTTCTCACCTTCTGGGATCCAGAG(SEQ ID NO.8);

[0450] EGFR-del1-S:

[0451] CTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAAACATCTCCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTG(SEQ ID NO.9);

[0452] EGFR-del1-AS:

[0453] CAGCAAAGCAGAAACTCACATCGAGGATTTCCTTGTTGGCTTTCGGAGATGTTTTGATAGCGACGGGAATTTTAACTTTCTCACCTTCTGGGATCCAGAG(SEQ ID NO.10);

[0454] EGFR-del2-S:

[0455] CTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGACATCTCCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTG(SEQ ID NO.11);

[0456] EGFR-del2-AS:

[0457] CAGCAAAGCAGAAACTCACATCGAGGATTTCCTTGTTGGCTTTCGGAGATGTCTTGATAGCGACGGGAATTTTAACTTTCTCACCTTCTGGGATCCAGAG (SEQ ID NO. 12);

[0458] EGFR-del3-S:

[0459] TCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAATCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTG (SEQ ID NO. 13);

[0460] EGFR-del3-AS:

[0461] CACACAGCAAAGCAGAAACTCACATCGAGGATTTCCTTGTTGGCTTTCGATTCCTTGATAGCGACGGGAATTTTAACTTTCTCACCTTCTGGGATCCAGA (SEQ ID NO. 14);

[0462] Synthesized by Shanghai Shenggong (Biological) Co., Ltd.

[0463] The wild-type allele of EGFR 19del was derived from the B-CPAP cell line; the mutant alleles EGFR 19E746_A750 del (2235-2249del) and EGFR 19 E746_A750 del (2236-2250del) were derived from NCI-H1650 and HCC827 cell lines, respectively. The cell lines were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. The genomic DNA of the two cell lines was extracted using the QIAGENDNeasy Blood&Tissue Kit according to the instructions, and the two genomic DNAs were prepared into templates with mutation ratios MAF = 5%, 1%, 0.5%, 0.1% and 0.01% for mutation enrichment and detection.

[0464] 4. sgRNA cutting verification

[0465] a) Formation of ribonucleoprotein complex (ribonucleoprotein), see Table 1.

[0466] Table 1 Ribonucleoprotein complex

[0467] Final concentration Cas9 2.5μM sgRNA 2.5μM HEPES 1M

[0468] The Cas protein here includes one of SpCas9 HF1 (for enrichment and detection of ED1), EvoCas9 (for enrichment and detection of ED1), SpCas9 (for enrichment and detection of ED2), and Hypa Cas9 (for enrichment and detection of ED2).

[0469] Incubate at 37°C for 10 min.

[0470] b) Cutting system preparation, see Table 2.

[0471] Table 2 Cutting system

[0472] Final concentration Ribonucleoprotein complex 2.5μM MgOAc 14mM template 0.25μM

[0473] 37℃, constant temperature cutting for 1h;

[0474] c) Digestion

[0475] Add 1 μl RNaseA (10 mg / ml) and let it stand at room temperature for 10 min; add 1 μl ProteinaseK (20 mg / ml) and keep the temperature at 56°C for 30 min; then keep the temperature at 95°C for 10 min.

[0476] d) Glue running

[0477] Use PAGE gel electrophoresis, add loading buffer to the cleavage product, and spot it; electrophoresis 150V, 10min;

[0478] After electrophoresis, stain with gelred dye for 15 minutes and take pictures (see Figure 2 ). Figure 2 The five lanes from left to right are: Marker, WT, ED1, ED2, ED3, where ED1 represents E746_A750 del (2235-2249del) mutation, ED2 represents EGFR 19 E746_A750 del (2236-2250del) mutation, and ED3 represents EGFR 19 E746_A750 del (2240-2257del) mutation; Figure 2 It can be seen that under the guidance of sgRNA, the wild type was cut, while other mutant types were hardly cut.

[0479] 5. Enrichment and detection of mutant alleles

[0480] The mutations enriched and detected in this step are ED1 and ED2.

[0481] a) Pre-amplification:

[0482] The formation of ribonucleoprotein complex (ribonucleoprotein) is shown in Table 3.

[0483] Table 3 Ribonucleoprotein complex

[0484] Final concentration Cas9 1μM sgRNA 1μM HEPES 1M

[0485] The Cas protein here includes one of SpCas9 HF1 (for enrichment and detection of ED1), EvoCas9 (for enrichment and detection of ED1), SpCas9 (for enrichment and detection of ED2), and Hypa Cas9 (for enrichment and detection of ED2); in specific operations, the Cas protein used in this step to prepare the ribonucleoprotein complex corresponds to the Cas9 used in step 4 and sgRNA cutting verification;

[0486] Incubate at 37°C for 10 min to obtain the ribonucleoprotein complex;

[0487] The preparation of the pre-amplification system is shown in Table 4.

[0488] Table 4 Pre-amplification system

[0489] Final concentration Ribonucleoprotein complex 1μM Pre-amplification F primer 0.5μM Pre-amplification R primer 0.5μM MgOAc 14mM dNTP 0.45mM DNA polymerase 30ng / μl Recombinase 900ng / μl SSB 150ng / μl template 60ng

[0490] 37℃, constant temperature reaction for 20min; then 95℃, 10min to terminate the reaction;

[0491] Digestion:

[0492] Add 1 μl RNaseA (10 mg / ml) and let it stand at room temperature for 10 min; add 1 μl ProteinaseK (20 mg / ml) and keep it at 56°C for 30 min; then keep it at 95°C for 10 min;

[0493] The qPCR system preparation is shown in Table 5.

[0494] Table 5 qPCR system

[0495] Final concentration qPCR F primer 1μM qPCR R primers 1μM dNTP 0.45mM DNA polymerase 30ng / μl

[0496] Reaction procedure:

[0497] Pre-denaturation: 95°C, 5 min;

[0498] 45 cycles;

[0499] 95℃, 10s;

[0500] 60℃, 30s;

[0501] After amplification, the amplified product was subjected to Sanger sequencing. The sequencing results were as follows Figure 3-9As shown, to avoid redundancy, only the enrichment results of samples with MAF=0.1% are shown; Figure 3-9 It can be seen that the wild-type allele increased significantly after enrichment, indicating that the enrichment method of the present invention can effectively enrich mutant alleles.

[0502] Example 2

[0503] Enrichment and detection of BRAF gene V600E mutation

[0504] 1. Primer design

[0505] The human BRAF gene sequence was obtained from the NCBI database, and primers were designed based on the sequence as follows:

[0506] Preamplification F primer: CTACACCTCAGATATATTTC (SEQ ID NO. 19);

[0507] Pre-amplification R primer: TGGATCCAGACAACTGT (SEQ ID NO. 20);

[0508] qPCR F primer: CCTCAGATATATTTCTTCATGA (SEQ ID NO. 21);

[0509] qPCR R primer: TGTTCAAACTGATGGGAC (SEQ ID NO. 22).

[0510] 2. Design sgRNA

[0511] Design an sgRNA sequence with a length of 17-20 nt that can completely match the wild-type allele sequence, as shown in SEQ ID NO.2-6:

[0512] guide1:UAGCUACAGUGAACUCUCGA (SEQ ID NO.2);

[0513] guide2:UAGCUACAGUGAAAUCACGA (SEQ ID NO.3);

[0514] guide3:GCUACAGUGAACUCUCGA (SEQ ID NO.4);

[0515] guide4:GUCUAGCUACAGUGAAA (SEQ ID NO.5);

[0516] guide5:GUCUAGCUGCAGUGAAA (SEQ ID NO. 6).

[0517] 3. Genomic DNA acquisition

[0518] BRAF V600E mutant and wild-type alleles were derived from B-CPAP and HCC827 cell lines, respectively (purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.); genomic DNA of the two cell lines was extracted using QIAGEN DNeasy Blood&Tissue Kit according to the instructions, and the two DNAs were prepared into templates with mutation ratios MAF=5%, 1%, 0.5%, 0.1% and 0.01% for mutation enrichment and detection.

[0519] 4. sgRNA cutting verification

[0520] a) Formation of ribonucleoprotein complex

[0521] Prepare the following reaction system, see Table 6.

[0522] Table 6 Reaction system

[0523] Final concentration Cas9 2.5μM sgRNA 2.5μM HEPES 1M

[0524] Here, Cas9 includes one of HiFi Cas9, SuperFi Cas9, and HiFi SC++;

[0525] Incubate at 37°C for 10 min to obtain the ribonucleoprotein complex;

[0526] b) Cutting system preparation, see Table 7.

[0527] Table 7 Cutting system

[0528] Final concentration Ribonucleoprotein complex 2.5μM MgOAc 14mM template 0.25μM

[0529] 37℃, constant temperature cutting for 1h;

[0530] c) Digestion

[0531] Add 1 μl RNaseA (10 mg / ml) and let it stand at room temperature for 10 min; add 1 μl ProteinaseK (20 mg / ml) and keep it at 56°C for 30 min; then keep it at 95°C for 10 min;

[0532] d) Glue running

[0533] Use PAGE gel electrophoresis, add loading buffer to the cleavage product, and spot it; electrophoresis 150V, 10min;

[0534] After electrophoresis, stain with gelred dye for 15 minutes and take pictures (see Figure 7), where lanes 1, 3, 5, 7, 9, and 11 are wild-type nucleic acids, and lanes 2, 4, 6, 8, 10, and 12 are mutant nucleic acids; by comparing lanes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, it can be seen that the wild-type allele is more easily cut than the mutant allele; by comparing lanes 1, 3, 5, 7, 9, and 11, it can be seen that guide3 hardly cuts the wild-type allele.

[0535] 5. Enrichment and detection of mutant alleles

[0536] a) Pre-amplification:

[0537] The formation of ribonucleoprotein complex (ribonucleoprotein), see Table 8.

[0538] Table 8 Ribonucleoprotein complex

[0539] Final concentration Cas9 1μM sgRNA 1μM HEPES 1M

[0540] Incubate at 37°C for 10 min to obtain the ribonucleoprotein complex;

[0541] The preparation of the pre-amplification system is shown in Table 9.

[0542] Table 9 Pre-amplification system

[0543] Final concentration Ribonucleoprotein complex 1μM Pre-amplification F primer 0.5μM Pre-amplification R primer 0.5μM MgOAc 14mM dNTP 0.45mM DNA polymerase 30ng / μl Recombinase 900ng / μl SSB 150ng / μl template 60ng

[0544] Here, Cas9 includes: one of HiFi Cas9, SuperFi Cas9, and HiFi SC++; in specific operations, the Cas protein used in preparing the ribonucleoprotein complex in this step corresponds to the Cas9 used in step 4 and sgRNA cutting verification;

[0545] 37℃, constant temperature reaction for 20min; then 95℃, 10min to terminate the reaction;

[0546] Digestion:

[0547] Add 1 μl RNaseA (10 mg / ml) and let it stand at room temperature for 10 min; add 1 μl ProteinaseK (20 mg / ml) and keep it at 56°C for 30 min; then keep it at 95°C for 10 min;

[0548] For the preparation of qPCR system, see Table 10.

[0549] Table 10 qPCR system

[0550] Final concentration qPCR F primer 1μM qPCR R primers 1μM dNTP 0.45mM DNA polymerase 30ng / μl

[0551] Reaction procedure:

[0552] Pre-denaturation: 95°C, 5 min;

[0553] 45 cycles;

[0554] 95℃, 10s;

[0555] 60℃, 30s;

[0556] After amplification, the amplified product was subjected to Sanger sequencing. The sequencing results were as follows Figure 5 As shown, to avoid redundancy, only the enrichment results of samples with MAF=0.1% are shown; Figure 8-14 It can be seen that the wild-type allele increased significantly after enrichment; and, compared Figure 10-14 It can be seen that as the treatment time with Cas protein increases, the enrichment effect becomes significantly better, indicating that the enrichment method of the present invention can effectively enrich mutant alleles.

[0557] Example 3

[0558] Enrichment and detection of EGFR gene T790M mutation

[0559] 1. Primer design

[0560] The human EGFR gene sequence was obtained from the NCBI database, and primers were designed based on the sequence as follows:

[0561] Preamplification F primer: TACGTGATGGCCAGCGTGGA (SEQ ID NO. 23);

[0562] Pre-amplification R primer: ACTGGGAGCCAATATTGT (SEQ ID NO. 24);

[0563] qPCR F primer: TGATGGCCAGCGTGGACAA (SEQ ID NO. 25);

[0564] qPCR R primer: TTGTGTTCCCGGACATAGTC (SEQ ID NO. 26).

[0565] 2. Design sgRNA

[0566] Design sgRNA sequences with a length of 17-20 nt and that can completely match the wild-type allele sequence, as shown in SEQ ID NO.27-28:

[0567] guide1:GGCAGCCGAAGGGCAUGAGC (SEQ ID NO.27);

[0568] guide2:GGCAGCCGAAGAGCAUGAGC (SEQ ID NO.28);

[0569] 3. Acquisition of target genes

[0570] The following is the short gene sequence used as a marker in this example:

[0571] T790M-WT-S:

[0572] GACATAGTCCAGGAGGCAGCCGAAGGGCATGAGCTGCGTGATGAGCTGCACGGTGGAGGTGAGGCAGATGCCCAGCAGGC (SEQ ID NO. 29);

[0573] T790M-WT-AS:

[0574] GCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATCACGCAGCTCATGCCCTTCGGCTGCCTCCTGGACTATGTC (SEQ ID NO. 30);

[0575] T790M-MUT-S:

[0576] GACATAGTCCAGGAGGCAGCCGAAGGGCATGAGCTGCATGATGAGCTGCACGGTGGAGGTGAGGCAGATGCCCAGCAGGC (SEQ ID NO. 31);

[0577] T790M-MUT-AS:

[0578] GCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATCATGCAGCTCATGCCCTTCGGCTGCCTCCTGGACTATGTC (SEQ ID NO. 32);

[0579] Synthesized by Shanghai Shenggong (Biological) Co., Ltd.

[0580] Obtaining genomic DNA:

[0581] The EGFR T790M wild-type allele was derived from the B-CPAP cell line and purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. The genomic DNA was extracted using the QIAGEN DNeasy Blood & Tissue Kit. The EGFR T790M mutant allele sequence standard was purchased from GeneCopoeia. The genomic DNA and mutant allele sequence standard were prepared into templates with mutation ratios MAF = 5%, 1%, 0.5%, 0.1% and 0.01%.

[0582] 4. sgRNA cutting verification

[0583] a) Formation of ribonucleoprotein complex (ribonucleoprotein), see Table 11.

[0584] Table 11 Ribonucleoprotein complex

[0585] Final concentration Cas9 2.5μM sgRNA (guide2) 2.5μM HEPES 1M

[0586] Incubate at 37°C for 10 min.

[0587] b) Cutting system preparation, see Table 12.

[0588] Table 12 Cutting system

[0589] Final concentration Ribonucleoprotein complex 2.5μM MgOAc 14mM template 0.25μM

[0590] 37℃, constant temperature cutting for 1h;

[0591] c) Digestion

[0592] Add 1 μl RNaseA (10 mg / ml) and let it stand at room temperature for 10 min; add 1 μl ProteinaseK (20 mg / ml) and keep the temperature at 56°C for 30 min; then keep the temperature at 95°C for 10 min.

[0593] d) Glue running

[0594] Use PAGE gel electrophoresis, add loading buffer to the cleavage product, and spot it; electrophoresis 150V, 10min;

[0595] After electrophoresis, stain with gelred dye for 15 minutes and take pictures (see Fig.15 ). Fig.15 The two lanes in the middle are the wild type and mutant from left to right.

[0596] 5. Enrichment and detection of mutant alleles

[0597] a) Pre-amplification:

[0598] The formation of ribonucleoprotein complex (ribonucleoprotein), see Table 13.

[0599] Table 13 Ribonucleoprotein complex

[0600] Final concentration Cas9 1.4μM sgRNA 1.4μM HEPES 1M

[0601] Incubate at 37°C for 10 min to obtain the ribonucleoprotein complex;

[0602] The preparation of the pre-amplification system is shown in Table 14.

[0603] Table 14 Pre-amplification system

[0604] Final concentration Ribonucleoprotein complex 1μM Pre-amplification F primer 0.5μM Pre-amplification R primer 0.5μM MgOAc 14mM dNTP 0.45mM DNA polymerase 30ng / μl Recombinase 900ng / μl SSB 150ng / μl template 60ng

[0605] 37℃, constant temperature reaction for 20min; then 95℃, 10min to terminate the reaction;

[0606] Digestion:

[0607] Add 1 μl RNaseA (10 mg / ml) and let it stand at room temperature for 10 min; add 1 μl ProteinaseK (20 mg / ml) and keep it at 56°C for 30 min; then keep it at 95°C for 10 min;

[0608] For the preparation of qPCR system, see Table 15.

[0609] Table 15 qPCR system

[0610] Final concentration qPCR F primer 1μM qPCR R primers 1μM dNTP 0.45mM DNA polymerase 30ng / μl

[0611] Reaction procedure:

[0612] Pre-denaturation: 95°C, 5 min;

[0613] 45 cycles;

[0614] 95℃, 10s;

[0615] 60℃, 30s;

[0616] After amplification, the amplified product was subjected to Sanger sequencing. The sequencing results were as follows Fig.16 As shown, to avoid redundancy, only the enrichment results of samples with MAF=5% are shown; Fig.16 It can be seen that the wild-type allele increased significantly after enrichment, indicating that the enrichment method of the present invention can effectively enrich mutant alleles.

[0617] Example 4

[0618] Enrichment and detection of PCDH10 gene methylation

[0619] 1. Primer design

[0620] The human PCDH10 gene sequence was obtained from the NCBI database, and primers were designed based on the sequence as follows:

[0621] Preamplification F primer: TCGTTAAATAGATACGTTACGC (SEQ ID NO. 33);

[0622] Pre-amplification R primer: TAAAAAACTAAAAACTTTCCGCG (SEQ ID NO. 34);

[0623] qPCR F primer: TCGTTAAATAGATACGTTACGC (SEQ ID NO.35);

[0624] qPCR R primer: CAACGCCTCGAAACCTACG (SEQ ID NO. 36).

[0625] 2. Design sgRNA

[0626] Design an sgRNA sequence with a length of 17-20 nt that can completely match the wild-type allele sequence, as shown in SEQ ID NO.37:

[0627] guide:AAUUUUUGUUUGAGUGGUUG (SEQ ID NO.37);

[0628] 3. Acquisition of genomic DNA

[0629] A549 is a PCDH10 non-methylated cell line purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. Genomic DNA was extracted using QIAGEN DNeasy Blood & Tissue Kit. Methylated Human Control was purchased from Promega. Templates with methylation ratios MAF = 5%, 1%, 0.5%, 0.1% and 0.01% were prepared.

[0630] 4. Enrichment and detection of methylated DNA

[0631] a) Pre-amplification:

[0632] The formation of ribonucleoprotein complex (ribonucleoprotein), see Table 16.

[0633] Table 16 Composition of ribonucleoprotein complex

[0634] Final concentration Cas9 1μM sgRNA 1μM HEPES 1M

[0635] Incubate at 37°C for 10 min to obtain the ribonucleoprotein complex;

[0636] The preparation of the pre-amplification system is shown in Table 17.

[0637] Table 17 Pre-amplification system

[0638] Final concentration Ribonucleoprotein complex 1μM Pre-amplification F primer 0.5μM Pre-amplification R primer 0.5μM MgOAc 14mM dNTP 0.45mM DNA polymerase 30ng / μl Recombinase 900ng / μl SSB 150ng / μl template 60ng

[0639] 37℃, constant temperature reaction for 20min; then 95℃, 10min to terminate the reaction;

[0640] Digestion:

[0641] Add 1 μl RNaseA (10 mg / ml) and let it stand at room temperature for 10 min; add 1 μl ProteinaseK (20 mg / ml) and keep it at 56°C for 30 min; then keep it at 95°C for 10 min;

[0642] For the preparation of qPCR system, see Table 18.

[0643] Table 18 qPCR system

[0644] Final concentration qPCR F primer 1μM qPCR R primers 1μM dNTP 0.45mM DNA polymerase 30ng / μl

[0645] Reaction procedure:

[0646] Pre-denaturation: 95°C, 5 min;

[0647] 30 cycles;

[0648] 95℃, 10s;

[0649] 60℃, 30s;

[0650] 72℃, 30s.

[0651] After amplification, the amplified product was subjected to Sanger sequencing. The sequencing results were as follows Fig.17 As shown, to avoid redundancy, only the enrichment results of samples with MAF=0.1% are shown; Fig.17 It can be seen that the methylated DNA increased significantly after enrichment, indicating that the enrichment method of the present invention can effectively enrich the methylated DNA.

[0652] Example 5

[0653] Enrichment of NRAS gene Q61R mutation

[0654] 1. Primer design

[0655] The gene sequence of exon 61 of the human NRAS gene was obtained from the NCBI database, and primers were designed based on the sequence as follows:

[0656] Preamplification F primer: CCCCCAGGATTCTTACAGAAAACAAGTGGT (SEQ ID NO. 38);

[0657] Pre-amplification R primer: GCAAATACACAGAGGAAGCCTTCGCCTGTCCTC (SEQ ID NO. 39);

[0658] qPCR F primer: CAAGTGGTTATAGATGGTGA (SEQ ID NO.40);

[0659] qPCR R primer: CGCCTGTCCTCATGTATTGG (SEQ ID NO. 41).

[0660] 2. Design sgRNA

[0661] The crRNA sequence with a length of 20 nt and capable of completely pairing with the wild-type allele sequence is designed, as shown in SEQ ID NO.42:

[0662] UCCAGCUGUAUCCAGUAUGU (SEQ ID NO. 42);

[0663] Use crRNA to guide Cas protein to bind to the wild-type allele and specifically cut the wild-type allele without cutting the mutant allele. The principle is as follows Fig.18 shown.

[0664] 3. Acquisition of target genes

[0665] The wild-type allele of NRAS Q61R was derived from the IOSE80 cell line; the mutant allele of NRAS Q61R was derived from the SK-MEL-2 cell line. Both cell lines were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. The genomic DNA of the two cell lines was extracted using the QIAGENDNeasy Blood & Tissue Kit according to the instructions, and the two genomic DNAs were prepared into templates with mutation ratios MAF = 10%, 5%, 2.5%, 1%, 0.5%, 0.1%, 0.05%, 0.03% and 0.01% for mutation enrichment and detection.

[0666] 4. Enrichment and detection of mutant alleles

[0667] a) Pre-amplification:

[0668] The formation of ribonucleoprotein complex (ribonucleoprotein), see Table 19.

[0669] Table 19 Composition of ribonucleoprotein complex

[0670] Final concentration Cas12 10μM crRNA 10μM

[0671] Here, the Cas protein is FnCas12a, and the ribonucleoprotein complex is obtained by constant temperature incubation at 37°C for 10 min;

[0672] The preparation of the pre-amplification system is shown in Table 20.

[0673] Table 20 Pre-amplification system

[0674] Final concentration Ribonucleoprotein complex 10μM Pre-amplification F primer 0.48μM Pre-amplification R primer 0.48μM MgOAc 14mM dNTP 0.45mM DNA polymerase 30ng / μl Recombinase 900ng / μl SSB 150ng / μl template 60ng

[0675] 37℃, constant temperature reaction for 20min; then 95℃, 10min to terminate the reaction;

[0676] Digestion:

[0677] Add 1 μl RNaseA (10 mg / ml) and let it stand at room temperature for 10 min; add 1 μl ProteinaseK (20 mg / ml) and keep it at 56°C for 30 min; then keep it at 95°C for 10 min;

[0678] For qPCR system preparation, see Table 21.

[0679] Table 21 qPCR system

[0680] Final concentration qPCR F primer 1μM qPCR R primers 1μM dNTP 0.45mM DNA polymerase 30ng / μl

[0681] Reaction procedure:

[0682] Pre-denaturation: 95°C, 5 min;

[0683] 45 cycles;

[0684] 95℃, 10s;

[0685] 60℃, 30s;

[0686] After amplification, the amplified product was subjected to Sanger sequencing. The sequencing results were as follows Fig.19 As shown, to avoid redundancy, only the enrichment results of samples with MAF=0.05% are shown; Fig.19 It can be seen that the wild-type allele increased significantly after enrichment, indicating that the enrichment method of the present invention can effectively enrich mutant alleles.

[0687] Example 6

[0688] PASEA Automation

[0689] The enrichment and detection experiments performed in the above examples can be automated using a microfluidic chip. The microfluidic chip structure (see Fig. 20) is divided into three parts, consisting of a top encapsulation sheet 1, a bottom encapsulation sheet 3 and a middle reaction layer 2;

[0690] Top package sheet 1 (see Fig.21 ) The structure is: microfluidic chip mounting hole 11; microfluidic chip packaging positioning hole 12; microfluidic chip injection hole 13;

[0691] Intermediate reaction layer 2 (see Fig. 22 ) structure is: microfluidic chip packaging positioning hole 21; microfluidic chip mounting hole 22; pre-amplification reaction chamber 23; siphon valve 24; digestion reaction chamber 25; pre-distribution chamber 26; PCR reaction chamber 27; waste liquid chamber 28; capillary valve 29; gas passage 210.

[0692] Bottom package sheet 3 (see Fig.23 ) structure is: microfluidic chip mounting hole 31; microfluidic chip packaging positioning hole 32; RNaseA sample adding cavity 33; ProteinaseK sample adding cavity 34.

[0693] The specific steps are as follows:

[0694] 1) Sample addition: Add the pre-amplification system to the pre-amplification reaction chamber; add 5 μl RNaseA (10 mg / ml) to the RNaseA sample chamber; add 5 μl ProteinaseK (20 mg / ml) to the ProteinaseK sample chamber; add the qPCR system to the PCR reaction chamber.

[0695] 2) Sealing: Use a suitable sealing aluminum foil pressure-sensitive film to seal the sample inlet.

[0696] 3) Load and complete the experiment: Load the sealed microfluidic chip onto the centrifugal microfluidic platform.

[0697] Setup process:

[0698] a: 37°C, constant temperature reaction for 20 min (pre-amplification);

[0699] b: Rotation speed 3000 rpm duration 1 min (the pre-amplification solution is centrifuged into the digestion chamber during rotation, and the siphon valve is opened when it stops);

[0700] c: Leave without heating for 10 min (RNase A digestion);

[0701] d: 56°C for 30 min (Protease K digestion);

[0702] e: Speed ​​1000rpm Duration 1min (pre-allocation);

[0703] f: speed 3000 rpm duration 2 min (centrifuge the quantitative digested pre-amplification solution into the PCR chamber);

[0704] g: 95℃, 5min; 45 cycles: 95℃, 10s60℃, 30s (PCR reaction).

[0705] Example 7

[0706] Ago protein-based enrichment and detection of BRAF V600E mutations

[0707] 1. Primer design

[0708] The human BRAF gene sequence was obtained from the NCBI database, and primers were designed based on the sequence as follows:

[0709] Preamplification F primer: CTACACCTCAGATATATTTC (SEQ ID NO. 19);

[0710] Pre-amplification R primer: TGGATCCAGACAACTGT (SEQ ID NO. 20);

[0711] qPCR F primer: CCTCAGATATATTTCTTCATGA (SEQ ID NO. 21);

[0712] qPCR R primer: TGTTCAAACTGATGGGAC (SEQ ID NO. 22).

[0713] 2. Design guide DNA

[0714] A guide DNA sequence with a length of 16-20 nt and capable of completely matching with the wild-type allele sequence is designed, as shown in SEQ ID NO.43-44:

[0715] Forward guide: p-TAGATTTCACTGTAGC-3' (SEQ ID NO. 43);

[0716] Reverse guide: p-TTCTAGCTACAGTGAA -3' (SEQ ID NO. 44);

[0717] Use guide DNA to guide Ago protein to bind to the wild-type allele and specifically cut the wild-type allele without cutting the mutant allele. The principle is as follows Fig.23 shown.

[0718] 3. Genomic DNA acquisition

[0719] BRAF V600E mutant and wild-type alleles were derived from B-CPAP and HCC827 cell lines, respectively (purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.); genomic DNA of the two cell lines was extracted using QIAGEN DNeasy Blood&Tissue Kit according to the instructions, and the two DNAs were prepared into templates with mutation ratios MAF=5%, 1%, 0.5%, 0.1% and 0.01% for mutation enrichment and detection.

[0720] 5. Enrichment and detection of mutant alleles

[0721] a) Pre-amplification:

[0722] The formation of Ago-Guide DNA complex is shown in Table 22.

[0723] Table 22 Composition of Ago-Guide DNA complex

[0724] Final concentration Ago 1μM GuideDNA 10μM HEPES 1M

[0725] Incubate at 37°C for 10 min to obtain the ribonucleoprotein complex;

[0726] The preparation of the pre-amplification system is shown in Table 23.

[0727] Table 23 Pre-amplification system

[0728] Final concentration Ribonucleoprotein complex 1μM Pre-amplification F primer 0.5μM Pre-amplification R primer 0.5μM MgOAc 14mM dNTP 0.45mM DNA polymerase 30ng / μl Recombinase 900ng / μl SSB 150ng / μl template 60ng

[0729] Here, Ago includes one of CbAgo, TtAgo, KmAgo, and KpAgo;

[0730] 37℃, constant temperature reaction for 20min; then 95℃, 10min to terminate the reaction;

[0731] Digestion:

[0732] Add 1 μl RNaseA (10 mg / ml) and let it stand at room temperature for 10 min; add 1 μl ProteinaseK (20 mg / ml) and keep it at 56°C for 30 min; then keep it at 95°C for 10 min;

[0733] For qPCR system preparation, see Table 24.

[0734] Table 24 qPCR system

[0735] Final concentration qPCR F primer 1μM qPCR R primers 1μM dNTP 0.45mM DNA polymerase 30ng / μl

[0736] Reaction procedure:

[0737] Pre-denaturation: 95°C, 5 min;

[0738] 45 cycles;

[0739] 95℃, 10s;

[0740] 60℃, 30s;

[0741] After amplification, the amplified product was subjected to Sanger sequencing. The sequencing results were as follows Fig.25 As shown, to avoid redundancy, only the enrichment results of samples with MAF=0.1% are shown; Fig.25 It can be seen that the wild-type allele increased significantly after enrichment, indicating that the enrichment method of the present invention can also effectively enrich mutant alleles based on the specific cleavage of Ago protein.

[0742] The conventional operations in the operating steps of the present invention are well known to those skilled in the art and will not be described in detail here.

[0743] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A kit for enriching mutant genes, comprising: A ribonucleoprotein complex and a constant temperature cutting-amplification reaction system, wherein the constant temperature cutting-amplification reaction system is a cutting-while-amplification system, comprising: a primer, a target gene, an enzyme and a color-aiding group; The ribonucleoprotein complex comprises component (a) and component (b): Component (a) is sgRNA or crRNA, and component (b) is Cas protein; The mutant gene is the EGFR gene del19 mutation, the primers include 19del pre-amplification F primer, 19del pre-amplification R primer, 19del qPCR F primer and 19del qPCR R primer, 19del pre-amplification F primer is shown in SEQ ID NO.15, 19del pre-amplification R primer is shown in SEQ ID NO.16, 19del qPCR F primer is shown in SEQ ID NO.17, 19delqPCR R primer is shown in SEQ ID NO.18, the component (a) is sgRNA, the sequence is shown in SEQ ID NO.1, and the Cas protein is one of SpCas9 HF1, Evo Cas9, SpCas9 and Hypa Cas9; or, The mutant gene is the Q61R mutation of the NRAS gene, the primers include a Q61R preamplification F primer, a Q61R preamplification R primer, a Q61R qPCR F primer and a Q61R qPCR R primer, the Q61R preamplification F primer is shown in SEQ ID NO.38, the Q61R preamplification R primer is shown in SEQ ID NO.39, the Q61R qPCR F primer is shown in SEQ ID NO.40, the Q61R qPCR R primer is shown in SEQ ID NO.41, the component (a) is crRNA, the sequence is shown in SEQ ID NO.42, and the Cas protein is FnCas12a.

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