Ultra-sensitive target nucleic acid enrichment detection method based on programmable nuclease

Abstract

The invention discloses an ultra-sensitive target nucleic acid enrichment detection method based on programmable nuclease, and belongs to the technical field of biology. According to the enrichment method disclosed by the invention, on the basis of specific cutting of programmable nuclease, wild type nucleic acid can be continuously removed while isothermal amplification is carried out, so that the number of mutant alleles in a sample is continuously and exponentially increased, and low-abundance mutant genes or methylated DNA are enriched or detected. According to the present invention, sgRNA, guide DNA or guide RNA are designed and optimized, the sgRNA, the guide DNA or the guide RNA and a Cas protein or an Ago protein are used to form a ribonucleoprotein complex, the isothermal amplification is performed while the specific cutting is performed on the target gene, the mutation-containing gene is not cut, and the low-abundance mutation gene is enriched;

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to a super-sensitive target nucleic acid enrichment and detection method based on programmable nucleases. Background Art

[0002] Somatic mutations are closely related to tumorigenesis. Mutation detection is of great significance for early disease screening, precise treatment decision-making, 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, and has the advantages of non-invasiveness and easy implementation of dynamic monitoring. However, liquid biopsy faces the following problems: 1) Mutant nucleic acids and wild-type nucleic acids have high sequence homology, usually with only single or several nucleotide differences, which poses a challenge to identifying low-abundance mutant alleles among a large number of free wild-type alleles; 2) The detection of low-abundance methylated genes in body fluids also faces the same problem. Methylated genes and non-methylated genes have the same sequence, only with differences in base modification, which poses a challenge to identifying low-abundance methylated genes among a large number of free non-methylated genes.

[0003] Currently, high-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 mainly relies on ultra-deep (50000X) NGS sequencing, which has the problems of high cost, long time consumption, and difficulty in popularizing to the clinic. Therefore, there is an urgent need to develop a low-cost super-sensitive method for detecting extremely low-abundance mutant alleles to promote clinical application.

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

[0005] The cleavage of Cas protein is restricted by the PAM site. When mutations cause the disruption of the PAM site, in the same sample, the Cas protein targets and cleaves a large number of wild-type nucleic acids while retaining the mutant nucleic acids. Some 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 the PAM site. Argonaute protein is not restricted by the PAM site, and some researchers have developed the NAVIGATER (Nucleic Acid enrichment Via DNA Guided Argonaute from Thermusthermophilus) low-abundance mutant allele detection method.

[0006] Although the DASH method and CUT-PCR have improved the sensitivity of downstream analysis to some extent, the effectiveness of existing programmable endonuclease-based detection methods is affected by ineffective binding events between the enzyme and the target. Due to the very slow dissociation efficiency of Cas9, some wild-type allele targets are protected from cleavage, while non-specific off-target cleavage depletes extremely rare mutant alleles. A similar problem exists with NAVIGATER. To overcome these drawbacks, researchers have employed multiple rounds of selective cleavage of wild-type alleles followed by polymerase chain reaction to enrich mutant allele fragments 10-fold. Despite these improvements, high sensitivity for mutant allele detection still requires the use of next generation sequencing (NGS) or reliance on digital PCR, making these methods laborious, time-consuming, and expensive.

[0007] For the detection of low-abundance methylated genes in liquid biopsy, similar problems exist with the above techniques and it is also difficult to meet the requirements. Summary of the Invention

[0008] The object of the present invention is to provide a super-sensitive tumor-related gene enrichment method based on programmable nucleases. Using the method of the present invention, low-abundance mutant genes and methylated genes can be efficiently enriched, and at the same time, the method of the present invention can be automated by using microfluidic chip technology.

[0009] To achieve the above object of the invention, the following technical solutions are adopted: A ribonucleoprotein complex, comprising: Component (a), comprising a nucleic acid region capable of complementary base pairing with a target nucleic acid and a 0-4bp mismatch fragment; or a nucleic acid region capable of complementary base pairing with a target nucleic acid and an adjacent PAM including a mutation-prone site; Component (b), capable of binding to a target nucleic acid and cleaving the target nucleic acid strand; 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; component (a) and (b) are capable of binding. Preferably, component (a) can carry a label, and the label includes streptavidin or biotin.

[0010] Preferably, the length of the mismatched nucleotide fragment is 0bp or 1bp or 2bp or 3bp or 4bp.

[0011] Preferably, component (a) carries a chemical modification, and the chemical modification includes sulfur substitution for oxygen or methoxy modification.

[0012] Preferably, the sgRNA, guide DNA or guide RNA in component (a) includes a mismatched nucleotide fragment. The mismatched nucleotide fragment designed in the present invention is beneficial to expanding the use range of sgRNA, guide DNA or guide RNA, and can enable sgRNA, guide DNA or guide RNA to recognize more mutations.

[0013] Preferably, the PAM adjacent to the sgRNA in component (a) contains a tumor hot spot mutation site.

[0014] Preferably, the length of the spacer sequence of the sgRNA in component (a) is 16 - 22nt.

[0015] More preferably, the sgRNA sequence is as follows: UCUUAAUUCCUUGAUAGCGA (SEQ ID NO.1); UAGCUACAGUGAACUCUCGA (SEQ ID NO.2); UAGCUACAGUGAAAUCACGA (SEQ ID NO.3); GCUACAGUGAACUCUCGA (SEQ ID NO.4); GUCUAGCUACAGUGAAA (SEQ ID NO.5); GUCUAGCUGCAGUGAAA (SEQ ID NO.6); GGCAGCCGAAGGGCAUGAGC (SEQ ID NO.27); GGCAGCCGAAGAGCAUGAGC (SEQ ID NO.28); AAUUUUUGUUUGAGUGGUUG (SEQ ID NO.37); UCCAGCUGUAUCCAGUAUGU (SEQ ID NO.42).

[0016] More preferably, the guide DNA sequences are as follows: Forward guide: p-TAGATTTCACTGTAGC-3' (SEQ ID NO.43); Reverse guide: p-TTCTAGCTACAGTGAA -3' (SEQ ID NO.44).

[0017] 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.

[0018] More preferably, Cas12a is from at least one of the following strains: Francisella novicida; Acidaminococcus;Lachnospiraceae .

[0019] More preferably, SacCas9 is from the strain Staphylococcus aureus .

[0020] More preferably, CjCas9 is from the strain Campylobacter jejuni .

[0021] More preferably, SpCas9 is from the strain Streptococcus pyogenes .

[0022] More preferably, NmCas9 is from the strain Neisseria meningitidis .

[0023] More preferably, SaCas9 is from the strain Staphylococcus aureus .

[0024] More preferably, ScCas9 is from the strain Streptococcus canis .

[0025] More preferably, SpaCas9 is from the bacterial strain Streptococcus pasteurianus .

[0026] Preferably, in the ribonucleoprotein complex, the final concentration of component (a) is 0.1 - 2 μM.

[0027] Preferably, in the ribonucleoprotein complex, the final concentration of component (b) is 0.1 - 3 μM.

[0028] Preferably, the ribonucleoprotein complex further comprises a hydrogen ion buffer.

[0029] More preferably, in the ribonucleoprotein complex, the final concentration of the hydrogen ion buffer is 1 mM - 2 M.

[0030] The present invention also discloses the use of the above ribonucleoprotein in enriching and / or detecting mutant genes.

[0031] The present invention also discloses the use of the above ribonucleoprotein in enriching and / or detecting methylated DNA.

[0032] Preferably, the gene includes 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.

[0033] 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.

[0034] Preferably, the hydrogen ion buffer includes HEPES.

[0035] Preferably, in the preparation of the ribonucleoprotein complex, the final concentration of Cas9 used is 0.1 - 3 μM.

[0036] More preferably, in the preparation of the ribonucleoprotein complex, the final concentration of Cas9 used is 2.5 μM.

[0037] Preferably, in the preparation of the ribonucleoprotein complex, the final concentration of the sgRNA used is 0.1 - 2 μM.

[0038] More preferably, in the preparation of the ribonucleoprotein complex, the final concentration of the sgRNA used is 2.5 μM.

[0039] Preferably, in the preparation of the ribonucleoprotein complex, the final concentration of the HEPES used is 1 mM - 2 M.

[0040] Preferably, in the preparation of the ribonucleoprotein complex, the final concentration of the HEPES used is 1 M.

[0041] Preferably, in the preparation of the ribonucleoprotein complex, the reaction temperature is 34 - 39 °C and the reaction time is 3 - 18 min.

[0042] More preferably, in the preparation of the ribonucleoprotein complex, the reaction temperature is 37 °C and the reaction time is 10 min.

[0043] The present invention also discloses a reaction system for the enrichment of mutant genes for programmable enzymes and / or the enrichment of methylated DNA, and the reaction system comprises the above ribonucleoprotein complex.

[0044] Preferably, the ribonucleoprotein complex comprises: Component (a), comprising a nucleic acid region capable of complementary base pairing with a target nucleic acid and a 0 - 4 bp mismatch fragment; Component (b), capable of binding to the target nucleic acid and cleaving the target nucleic acid strand; 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) comprises Cas protein and / or Cas protein derivatives and / or Ago protein and / or Ago protein derivatives; component (a) and (b) can bind. Preferably, component (a) carries a label, and the label comprises streptavidin or biotin.

[0045] Preferably, the length of the mismatched nucleotide fragment is 0 bp or 1 bp or 2 bp or 3 bp or 4 bp.

[0046] More preferably, component (a) carries a chemical modification, and the chemical modification comprises sulfur substitution for oxygen or methoxy modification.

[0047] More preferably, the sgRNA, guide DNA or guide RNA in component (a) comprises a mismatched nucleotide fragment.

[0048] More preferably, the spacer sequence length of the sgRNA, guide DNA or guide RNA in component (a) is 16-22 nt.

[0049] Even more preferably, the sgRNA sequence is as follows: UCUUAAUUCCUUGAUAGCGA (SEQ ID NO.1); UAGCUACAGUGAACUCUCGA (SEQ ID NO.2); UAGCUACAGUGAAAUCACGA (SEQ ID NO.3); GCUACAGUGAACUCUCGA (SEQ ID NO.4); GUCUAGCUACAGUGAAA (SEQ ID NO.5); GUCUAGCUGCAGUGAAA (SEQ ID NO.6); GGCAGCCGAAGGGCAUGAGC (SEQ ID NO.27); GGCAGCCGAAGAGCAUGAGC (SEQ ID NO.28); AAUUUUUGUUUGAGUGGUUG (SEQ ID NO.37); UCCAGCUGUAUCCAGUAUGU (SEQ ID NO.42).

[0050] 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.

[0051] Even more preferably, Cas12a is from at least one of the following strains: Francisella novicida; Acidaminococcus;Lachnospiraceae .

[0052] Even more preferably, SacCas9 is from the strain Staphylococcus aureus .

[0053] More preferably, CjCas9 is derived from the bacterial strain Campylobacter jejuni .

[0054] More preferably, SpCas9 is derived from the bacterial strain Streptococcus pyogenes .

[0055] More preferably, NmCas9 is derived from the bacterial strain Neisseria meningitidis .

[0056] Even more preferably, SaCas9 is derived from the bacterial strain Staphylococcus aureus .

[0057] Even more preferably, ScCas9 is derived from the bacterial strain Streptococcus canis .

[0058] Even more preferably, SpaCas9 is derived from the bacterial strain Streptococcus pasteurianus .

[0059] Even more preferably, in the ribonucleoprotein complex, the final concentration of component (a) is 0.1 - 2 μM.

[0060] Even more preferably, in the ribonucleoprotein complex, the final concentration of component (b) is 0.1 - 3 μM.

[0061] Even more preferably, the ribonucleoprotein complex further comprises a hydrogen ion buffer.

[0062] More preferably, in the ribonucleoprotein complex, the final concentration of the hydrogen ion buffer is 1 mM - 2 M.

[0063] Preferably, in the reaction system, the final concentration of the ribonucleoprotein complex is 0.1 - 2 μM.

[0064] Preferably, the reaction system further comprises: primers, a target gene, an enzyme, and a chromogenic group.

[0065] Preferably, the reaction system further comprises: dNTP, single-strand binding protein.

[0066] Even more preferably, the target gene comprises: genomic DNA or cell-free DNA.

[0067] More preferably, the target gene comprises genomic DNA extracted from at least one of the following cell lines: EGFR 19del wild-type cell line; EGFR 19 E746_A750 del (2235 - 2249del) cell line; EGFR19 E746_A750 del (2236 - 2250del); BRAF V600E mutant; BRAF V600E wild-type cell line; B-CPAP cell line.

[0068] More preferably, the primer includes a nucleotide sequence capable of binding to the target gene and guiding synthesis.

[0069] Even more preferably, the primer includes at least one of the following sequences: GCATGTGGCACCATCTCACA (SEQ ID NO.15); AGAGCAGCTGCCAGACATGA (SEQ ID NO.16); CTACACCTCAGATATATTTC (SEQ ID NO.19); TGGATCCAGACAACTGT (SEQ ID NO.20); TACGTGATGGCCAGCGTGGA (SEQ ID NO.23); ACTGGGAGCCAATATTGT (SEQ ID NO.24); TCGTTAAATAGATACGTTACGC (SEQ ID NO.33); TAAAAACTAAAAACTTTCCGCG (SEQ ID NO.34); TCGTTAAATAGATACGTTACGC (SEQ ID NO.35); CAACGCCTCGAAACCTACG (SEQ ID NO.36).

[0070] CCCCCAGGATTCTTACAGAAAACAAGTGGT (SEQ ID NO.38); GCAAATACACAGAGGAAGCCTTCGCCTGTCCTC (SEQ ID NO.39); CAAGTGGTTATAGATGGTGA (SEQ ID NO.40); CGCCTGTCCTCATGTATTGG (SEQ ID NO.41).

[0071] More preferably, the enzyme includes: DNA polymerase and / or recombinase.

[0072] More preferably, the chromophore group includes MgOAc.

[0073] Preferably, the above reaction system can be used to enrich mutant alleles with MAF ≥ 0.01%.

[0074] The present invention also discloses the uses of the above reaction system, including at least one of the following: (1) Targeted binding to a target nucleic acid fragment; (2) Targeted cleavage of a target nucleic acid fragment; (3) Enrichment of mutant genes; (4) Detection of mutant genes; (5) Enrichment of methylated DNA; or, (6) Detection of methylated DNA.

[0075] Preferably, the above enrichment includes automated enrichment; the above detection includes automated detection.

[0076] More preferably, the above automated enrichment includes enrichment using a microfluidic chip; the above automated detection includes detection using a microfluidic chip.

[0077] Even more preferably, the microfluidic chip is divided into three parts, consisting of a top encapsulation sheet, a bottom encapsulation sheet, and a middle reaction layer.

[0078] Even more preferably, the structure of the top encapsulation sheet includes: a microfluidic chip mounting hole; a microfluidic chip encapsulation positioning hole; a microfluidic chip sampling hole.

[0079] Even more preferably, the structure of the middle reaction layer includes: a microfluidic chip encapsulation positioning hole; a microfluidic chip mounting hole; a pre-amplification reaction chamber; a siphon valve; a digestion reaction chamber; a pre-distribution chamber; a PCR reaction chamber; a waste liquid chamber; a capillary valve; 10: a gas passage.

[0080] Even more preferably, the structure of the bottom encapsulation sheet includes: a microfluidic chip mounting hole; a microfluidic chip encapsulation positioning hole; an RNaseA sampling chamber; a ProteinaseK sampling chamber.

[0081] Preferably, the mutant genes include cancer-related genes.

[0082] Preferably, the cancer-related 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.

[0083] Preferably, the methylated DNA includes cancer-related methylated genes and / or their promoters.

[0084] Preferably, the cancer-related methylated genes include at least one of the following: PCDH-10, BRCA1, RASSF1A, ESR1, APC, p14ARF, p16INK4a, DAPK, CDH1, RUNX3, TFPI2, SFRP5, HIC1, PAX5, PGR, THBS1, ESR, COL23A1, C2CD4D, WNT6, OPCML, ZNF154, RARb2, ATM, MGMT, GSTP1, MIR129-2, LINC01158, CCDC181, PRKCB, TBR1, ZNF781, MARCH11, VWC2, SLC9A3, HOXA7, Septin9, IKZF1, BCAT1, hMLH1, WIF1, CDKN2A, SHOX2, 3OST2, ASSF1A, RARb, PITX2, NID2, NEUROG2 or HOXA1; The promoters include at least one of the promoters of the following genes: PCDH-10, BRCA1, RASSF1A, ESR1, APC, p14ARF, p16INK4a, DAPK, CDH1, RUNX3, TFPI2, SFRP5, HIC1, PAX5, PGR, THBS1, ESR, COL23A1, C2CD4D, WNT6, OPCML, ZNF154, RARb2, ATM, MGMT, GSTP1, MIR129-2, LINC01158, CCDC181, PRKCB, TBR1, ZNF781, MARCH11, VWC2, SLC9A3, HOXA7, Septin9, IKZF1, BCAT1, hMLH1, WIF1, CDKN2A, SHOX2, 3OST2, ASSF1A, RARb, PITX2, NID2, NEUROG2 or HOXA1.

[0085] The present invention also discloses the uses of the above ribonucleoprotein complex, including at least one of the following: (1) Enriching gene mutations; (2) Detecting gene mutations; (3) Enriching methylated DNA; or, (4) Detecting methylated DNA.

[0086] 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.

[0087] The present invention also discloses the uses of Cas proteins, including at least one of the following: (1) Enriching gene mutations; (2) Detecting gene mutations; (3) Enriching methylated DNA; or, (4) Detecting methylated DNA.

[0088] The present invention also discloses the uses of Ago proteins, including at least one of the following: (1) Enriching gene mutations; (2) Detecting gene mutations; (3) Enriching methylated DNA; or, (4) Detecting methylated DNA.

[0089] 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.

[0090] 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.

[0091] The present invention also discloses a kit for enriching mutant genes of programmable enzymes, and the kit includes the above ribonucleoprotein complex.

[0092] Preferably, the ribonucleoprotein complex includes: Component (a), comprising a nucleic acid region capable of complementary base pairing with the target nucleic acid and a 0-4bp mismatch fragment; Component (b), capable of binding to the target nucleic acid and breaking the target nucleic acid strand; 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; components (a) and (b) can bind.

[0093] Preferably, the length of the mismatched nucleotide fragment is 0bp or 1bp or 2bp or 3bp or 4bp.

[0094] Preferably, component (a) carries a label, and the label includes streptavidin or biotin.

[0095] More preferably, component (a) carries a chemical modification, and the chemical modification includes sulfur substitution for oxygen or methoxy modification.

[0096] More preferably, the sgRNA in component (a) includes a mismatched nucleotide fragment.

[0097] More preferably, the spacer sequence of the sgRNA in component (a) has a length of 16-22nt.

[0098] Even more preferably, the sgRNA sequence is as follows: UCUUAAUUCCUUGAUAGCGA (SEQ ID NO.1); UAGCUACAGUGAACUCUCGA (SEQ ID NO.2); UAGCUACAGUGAAAUCACGA (SEQ ID NO.3); GCUACAGUGAACUCUCGA (SEQ ID NO.4); GUCUAGCUACAGUGAAA (SEQ ID NO.5); GUCUAGCUGCAGUGAAA (SEQ ID NO.6); GGCAGCCGAAGGGCAUGAGC (SEQ ID NO.27); GGCAGCCGAAGAGCAUGAGC (SEQ ID NO.28); AAUUUUUGUUUGAGUGGUUG (SEQ ID NO.37); UCCAGCUGUAUCCAGUAUGU (SEQ ID NO.42).

[0099] More preferably, component (b) comprises 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.

[0100] Even more preferably, Cas12a is from at least one of the following strains: Francisella novicida; Acidaminococcus; Lachnospiraceae.

[0101] Even more preferably, SacCas9 is from the strain Staphylococcus aureus.

[0102] Even more preferably, CjCas9 is from the strain Campylobacter jejuni.

[0103] More preferably, SpCas9 is derived from the strain Streptococcus pyogenes.

[0104] More preferably, NmCas9 is derived from the strain Neisseria meningitidis.

[0105] More preferably, SaCas9 is derived from the strain Staphylococcus aureus.

[0106] More preferably, ScCas9 is derived from the strain Streptococcus canis.

[0107] More preferably, SpaCas9 is derived from the strain Streptococcus pasteurianus.

[0108] More preferably, in the ribonucleoprotein complex, the final concentration of component (a) is 0.1 - 2 μM.

[0109] More preferably, in the ribonucleoprotein complex, the final concentration of component (b) is 0.1 - 3 μM.

[0110] More preferably, the ribonucleoprotein complex further includes a hydrogen ion buffer.

[0111] More preferably, in the ribonucleoprotein complex, the final concentration of the hydrogen ion buffer is 1 mM - 2 M.

[0112] Preferably, the kit further includes a thermostatic cleavage - amplification reaction system.

[0113] Preferably, in the reaction system, the final concentration of the ribonucleoprotein complex is 0.1 - 2 μM.

[0114] Preferably, the reaction system further includes: primers, target genes, enzymes, and chromogenic groups.

[0115] Preferably, the reaction system further includes: dNTP, single - strand binding protein.

[0116] More preferably, the target gene includes: genomic DNA or cell - free DNA.

[0117] More preferably, the target gene includes genomic DNA extracted from at least one of the following cell lines: EGFR 19del wild-type cell line; EGFR 19 E746_A750 del (2235-2249del) cell line; EGFR19 E746_A750 del (2236-2250del); BRAF V600E mutant; BRAF V600E wild-type cell line; B-CPAP cell line.

[0118] More preferably, the primer comprises a nucleotide sequence capable of binding to a target gene and guiding synthesis.

[0119] Even more preferably, the primer comprises at least one of the following sequences: GCATGTGGCACCATCTCACA (SEQ ID NO.15); AGAGCAGCTGCCAGACATGA (SEQ ID NO.16); CTACACCTCAGATATATTTC (SEQ ID NO.19); TGGATCCAGACAACTGT (SEQ ID NO.20); TACGTGATGGCCAGCGTGGA (SEQ ID NO.23); ACTGGGAGCCAATATTGT (SEQ ID NO.24); TCGTTAAATAGATACGTTACGC (SEQ ID NO.33); TAAAAACTAAAAACTTTCCGCG (SEQ ID NO.34); TCGTTAAATAGATACGTTACGC (SEQ ID NO.35); CAACGCCTCGAAACCTACG (SEQ ID NO.36).

[0120] CCCCCAGGATTCTTACAGAAAACAAGTGGT (SEQ ID NO.38); GCAAATACACAGAGGAAGCCTTCGCCTGTCCTC (SEQ ID NO.39); CAAGTGGTTATAGATGGTGA (SEQ ID NO.40); or, CGCCTGTCCTCATGTATTGG (SEQ ID NO.41).

[0121] More preferably, the enzyme comprises: DNA polymerase and / or recombinase.

[0122] More preferably, the auxochrome group includes MgOAc.

[0123] Preferably, the above reaction system can be used to enrich mutant alleles with MAF ≥ 0.01%.

[0124] The present invention also discloses a method for detecting and / or enriching low-abundance mutant genes using the above kit, and the operation steps include: Collecting a sample; Preparing a ribonucleoprotein complex; Formulating a reaction system; Enrichment analysis.

[0125] Preferably, the step of collecting a sample includes extracting DNA and / or RNA in the sample using a nucleic acid extraction kit.

[0126] More preferably, the sample includes at least one of the following: blood, plasma / serum, cerebrospinal fluid, urine, saliva.

[0127] Even more preferably, the sample includes at least one of the following obtained from cancer patients: blood, plasma / serum, cerebrospinal fluid, urine, saliva.

[0128] Preferably, the step of preparing a ribonucleoprotein complex includes: Mixing and incubating Cas9, sgRNA, and a hydrogen ion buffer.

[0129] Preferably, the hydrogen ion buffer includes HEPES.

[0130] Preferably, in the preparation of the ribonucleoprotein complex, the final concentration of Cas9 used is 0.1 - 3 μM.

[0131] More preferably, in the preparation of the ribonucleoprotein complex, the final concentration of Cas9 used is 2.5 μM.

[0132] Preferably, in the preparation of the ribonucleoprotein complex, the final concentration of sgRNA used is 0.1 - 2 μM.

[0133] More preferably, in the preparation of the ribonucleoprotein complex, the final concentration of sgRNA used is 2.5 μM.

[0134] Preferably, in the preparation of the ribonucleoprotein complex, the final concentration of HEPES used is 1 mM - 2 M.

[0135] More preferably, in the preparation of the ribonucleoprotein complex, the final concentration of HEPES used is 1 M.

[0136] Preferably, in the preparation of the ribonucleoprotein complex, the reaction temperature is 34 - 39 °C, and the reaction time is 3 - 18 min.

[0137] More preferably, in the preparation of the ribonucleoprotein complex, the reaction temperature is 37 °C, and the reaction time is 10 min.

[0138] Preferably, the step of preparing the reaction system includes preparing a constant-temperature cleavage-amplification reaction system.

[0139] More preferably, in the reaction system, the final concentration of the ribonucleoprotein complex is 0.1 - 2 μM.

[0140] More preferably, the reaction system further includes: primers, target genes, enzymes, and chromogenic groups.

[0141] More preferably, the reaction system further includes: dNTP, single-stranded binding protein.

[0142] Even more preferably, the target gene includes: genomic DNA.

[0143] Even more preferably, the target gene includes genomic DNA extracted from at least one of the following cell lines: EGFR 19del wild-type cell line; EGFR 19 E746_A750 del (2235 - 2249del) cell line; EGFR19 E746_A750 del (2236 - 2250del); BRAF V600E mutant; BRAF V600E wild-type cell line; B-CPAP cell line.

[0144] Even more preferably, the primers include nucleotide sequences that can bind to the target gene and guide synthesis.

[0145] Even more preferably, the primers include at least one of the following sequences: GCATGTGGCACCATCTCACA (SEQ ID NO.15); AGAGCAGCTGCCAGACATGA (SEQ ID NO.16); CTACACCTCAGATATATTTC (SEQ ID NO.19); TGGATCCAGACAACTGT (SEQ ID NO.20); TACGTGATGGCCAGCGTGGA (SEQ ID NO.23); ACTGGGAGCCAATATTGT (SEQ ID NO.24); TCGTTAAATAGATACGTTACGC (SEQ ID NO.33); TAAAAACTAAAAACTTTCCGCG (SEQ ID NO.34); TCGTTAAATAGATACGTTACGC (SEQ ID NO.35); CAACGCCTCGAAACCTACG (SEQ ID NO.36).

[0146] CCCCCAGGATTCTTACAGAAAACAAGTGGT (SEQ ID NO.38); GCAAATACACAGAGGAAGCCTTCGCCTGTCCTC (SEQ ID NO.39); CAAGTGGTTATAGATGGTGA (SEQ ID NO.40); or, CGCCTGTCCTCATGTATTGG (SEQ ID NO.41).

[0147] More preferably, the enzyme includes: DNA polymerase and / or recombinase.

[0148] More preferably, the chromophore includes MgOAc.

[0149] Preferably, the enrichment analysis step includes QPCR analysis, sequencing, point-of-care testing (POCT), and the detection method for low-abundance mutant genes based on programmable nucleases as described above.

[0150] More preferably, the QPCR analysis step includes preparing a QPCR reaction system.

[0151] Even more preferably, the QPCR reaction system includes: Primers, chromophore, dNTP, enzyme.

[0152] Even more preferably, the primers include at least one of the following: TGTCATAGGGACTCTGGATCCCAGA (SEQ ID NO.17); GCAGAAACTCACATCGAGGATTTCCTTGT (SEQ ID NO.18); CCTCAGATATATTTCTTCATGA (SEQ ID NO.21); TGTTCAAACTGATGGGAC (SEQ ID NO.22); TGATGGCCAGCGTGGACAA (SEQ ID NO.25); or, TTGTGTTCCCGGACATAGTC (SEQ ID NO.26).

[0153] More preferably, the chromophore group includes MgOAc.

[0154] More preferably, the enzyme includes DNA polymerase.

[0155] The present invention also discloses a design method of sgRNA based on the CRISPR system, and the sgRNA includes a specific recognition nucleotide fragment and a mismatched nucleotide fragment; wherein, the length of the mismatched nucleotide fragment is 0 - 4bp.

[0156] Preferably, the specific recognition nucleotide fragment is a spacer sequence.

[0157] More preferably, the length of the spacer sequence is 18 - 22nt.

[0158] Preferably, the length of the mismatched nucleotide fragment is 0bp or 1bp or 2bp or 3bp or 4bp.

[0159] The sgRNA designed by using the sgRNA design method of the present invention can effectively distinguish between wild - type alleles and mutant alleles, and the spacer sequence of the sgRNA designed by using the sgRNA design method of the present invention has high recognition and cleavage efficiency. The mismatched nucleotide fragments designed by the present invention are beneficial to expanding the application range of sgRNA and can enable sgRNA to recognize more mutations.

[0160] The present invention also discloses an sgRNA, which includes a specific recognition nucleotide fragment and a mismatched nucleotide fragment; wherein, the length of the mismatched nucleotide fragment is 0 - 4bp.

[0161] Preferably, the length of the mismatched nucleotide fragment is 0bp or 1bp or 2bp or 3bp or 4bp.

[0162] Preferably, the above - mentioned sgRNA is designed by using the above - mentioned method.

[0163] Preferably, the site of the mismatched nucleotide is within the first 5bp and / or the last 5bp of the mutant gene.

[0164] Preferably, the specific recognition nucleotide fragment is a spacer sequence.

[0165] More preferably, the length of the spacer sequence is 18 - 22nt. [[ID=D43]]

[0166] More preferably, the sgRNA sequence is as follows: UCUUAAUUCCUUGAUAGCGA (SEQ ID NO.1); UAGCUACAGUGAACUCUCGA (SEQ ID NO.2); UAGCUACAGUGAAAUCACGA (SEQ ID NO.3); GCUACAGUGAACUCUCGA (SEQ ID NO.4); GUCUAGCUACAGUGAAA (SEQ ID NO.5); GUCUAGCUGCAGUGAAA (SEQ ID NO.6); GGCAGCCGAAGGGCAUGAGC (SEQ ID NO.27); GGCAGCCGAAGAGCAUGAGC (SEQ ID NO.28); AAUUUUUGUUUGAGUGGUUG (SEQ ID NO.37); UCCAGCUGUAUCCAGUAUGU (SEQ ID NO.42).

[0167] The present invention also discloses the uses of the above-mentioned sgRNAs, including at least one of the following: (1) Enriching gene mutations; (2) Detecting gene mutations; (3) Enriching methylated DNA; or, (4) Detecting methylated DNA.

[0168] The present invention also discloses a guide DNA or guide RNA, characterized in that the guide DNA or guide RNA comprises a specifically recognizing nucleotide fragment and a mismatched nucleotide fragment; wherein, the length of the mismatched nucleotide fragment is 0 - 4 bp.

[0169] Preferably, the length of the mismatched nucleotide fragment is 0 bp or 1 bp or 2 bp or 3 bp or 4 bp.

[0170] The present invention also discloses the uses of the above-mentioned guide DNA or guide RNA, including at least one of the following: (1) Enriching gene mutations; (2) Detecting gene mutations; (3) Enriching methylated DNA; or, (4) Detecting methylated DNA.

[0171] The present invention also discloses a method for enriching mutant genes and / or methylated DNA based on programmable enzymes, which uses the CRISPR system to specifically cleave wild-type alleles while amplifying mutant alleles; wherein, the CRISPR system includes: Component (a), comprising a nucleic acid region capable of complementary base pairing with a target nucleic acid and a 0-4bp mismatch fragment; Component (b), capable of binding to the target nucleic acid and breaking the target nucleic acid strand; 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; component (a) and (b) can bind. Preferably, component (a) carries a label, and the label includes streptavidin or biotin.

[0172] Preferably, the length of the mismatched nucleotide fragment is 0bp or 1bp or 2bp or 3bp or 4bp.

[0173] Preferably, component (a) carries a chemical modification, and the chemical modification includes sulfur substitution for oxygen or methoxy modification.

[0174] Preferably, the sgRNA in component (a) includes a mismatched nucleotide fragment. The mismatched nucleotide fragment designed in the present invention is beneficial to expanding the use range of sgRNA and enables sgRNA to recognize more mutations.

[0175] Preferably, the spacer sequence length of the sgRNA in component (a) is 16-22nt.

[0176] 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.

[0177] More preferably, Cas12a is from at least one of the following strains: Francisella novicida; Acidaminococcus; Lachnospiraceae.

[0178] More preferably, SacCas9 is from the strain Staphylococcus aureus.

[0179] More preferably, CjCas9 is from the strain Campylobacter jejuni.

[0180] More preferably, SpCas9 is from the strain Streptococcus pyogenes.

[0181] More preferably, NmCas9 is from the strain Neisseria meningitidis.

[0182] More preferably, SaCas9 is from the strain Staphylococcus aureus.

[0183] More preferably, ScCas9 is from the strain Streptococcus canis.

[0184] More preferably, SpaCas9 is from the strain Streptococcus pasteurianus.

[0185] Preferably, in the CRISPR system, the final concentration of component (a) is 0.1 - 2 μM.

[0186] Preferably, in the CRISPR system, the final concentration of component (b) is 0.1 - 3 μM.

[0187] Preferably, the CRISPR system further includes a hydrogen ion buffer.

[0188] More preferably, in the CRISPR system, the hydrogen ion buffer includes: HEPES.

[0189] More preferably, in the CRISPR system, the final concentration of the hydrogen ion buffer is 1 mM - 2 M.

[0190] Preferably, the methods for amplifying mutant alleles include PCR, LAMP, RPA.

[0191] More preferably, the reaction system for amplifying mutant alleles includes a ribonucleoprotein complex.

[0192] Preferably, in the reaction system, the final concentration of the ribonucleoprotein complex is 0.1 - 2 μM.

[0193] Preferably, the reaction system further comprises: primers, target genes, enzymes and auxochromic groups.

[0194] Preferably, the reaction system further comprises: dNTP and single-stranded binding protein.

[0195] More preferably, the target gene includes: genomic DNA.

[0196] More preferably, the target gene comprises genomic DNA extracted from at least one of the following cell lines: EGFR 19del wild-type cell line; EGFR 19 E746_A750 del (2235-2249del) cell line; EGFR19 E746_A750 del (2236-2250del); BRAF V600E mutant; BRAF V600E wild-type cell line; B-CPAP cell line.

[0197] More preferably, the primer comprises a nucleotide sequence capable of binding to the gene of interest and directing synthesis.

[0198] More preferably, the primer comprises at least one of the following sequences: GCATGTGGCACCATCTCACA (SEQ ID NO. 15); AGAGCAGCTGCCAGACATGA (SEQ ID NO. 16); CTACACCTCAGATATATTTC(SEQ ID NO.19); TGGATCCAGACAACTGT(SEQ ID NO.20); TACGTGATGGCCAGCGTGGA (SEQ ID NO. 23); ACTGGGAGCCAATATTGT(SEQ ID NO.24); TCGTTAAATAGATACGTTACGC (SEQ ID NO. 33); TAAAAACTAAAAACTTTCCCGG (SEQ ID NO. 34); TCGTTAAATAGATACGTTACGC (SEQ ID NO. 35); CAACGCCTCGAAACCTACG (SEQ ID NO. 36).

[0199] CCCCCAGGATTCTTACAGAAAACAAGTGGT (SEQ ID NO. 38); GCAAATACACAGAGGAAGCCTTCGCCTGTCCTC (SEQ ID NO.39); CAAGTGGTTATAGATGGTGA (SEQ ID NO.40); or, CGCCTGTCCTCATGTATTGG (SEQ ID NO.41).

[0200] More preferably, the enzyme includes: DNA polymerase and / or recombinase.

[0201] More preferably, the chromophore group includes MgOAc.

[0202] Even more preferably, the ribonucleoprotein complex includes Cas9, sgRNA and a hydrogen ion buffer.

[0203] Even more preferably, the method for preparing the ribonucleoprotein complex includes: mixing and incubating Cas9, sgRNA and a hydrogen ion buffer.

[0204] Even more preferably, the hydrogen ion buffer includes HEPES.

[0205] Even more preferably, in the preparation of the ribonucleoprotein complex, the final concentration of Cas9 used is 0.1 - 3 μM.

[0206] Even more preferably, in the preparation of the ribonucleoprotein complex, the final concentration of sgRNA used is 0.1 - 2 μM.

[0207] Even more preferably, in the preparation of the ribonucleoprotein complex, the final concentration of HEPES used is 1 mM - 2 M.

[0208] Even more preferably, in the preparation of the ribonucleoprotein complex, the reaction temperature is 34 - 39 °C and the reaction time is 3 - 18 min.

[0209] The present invention also discloses a method for detecting low-abundance mutant genes and / or methylated DNA based on programmable nucleases, which specifically cleaves wild-type alleles using the above CRISPR system while amplifying mutant alleles; it includes the following steps: Design sgRNA; Obtaining of the target gene; Amplification and cleavage; Detection of mutant genes.

[0210] Preferably, the sgRNA includes a mismatched nucleotide fragment.

[0211] Preferably, the length of the spacer sequence of the sgRNA is 16 - 22 nt.

[0212] 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.

[0213] Preferably, the amplification methods include: PCR, LAMP and RPA.

[0214] Preferably, the mutant gene detection method includes Sanger sequencing.

[0215] Preferably, the reaction system used for amplification includes: target gene, ribonucleoprotein complex, MgOAc, primer, DNA polymerase, recombinase and single-stranded binding protein.

[0216] Preferably, the reaction system used for cleavage includes: target gene, ribonucleoprotein complex, MgOAc.

[0217] The present invention also discloses a microfluidic chip for automatically enriching and / or detecting mutant genes and / or methylated DNA.

[0218] Preferably, the microfluidic chip is divided into three parts, consisting of a top encapsulation sheet, a bottom encapsulation sheet and an intermediate reaction layer.

[0219] More preferably, the structure of the top encapsulation sheet includes: a microfluidic chip mounting hole; a microfluidic chip encapsulation positioning hole; a microfluidic chip sampling hole.

[0220] More preferably, the structure of the intermediate reaction layer includes: a microfluidic chip encapsulation positioning hole; a microfluidic chip mounting hole; a pre-amplification reaction chamber; a siphon valve; a digestion reaction chamber; a pre-distribution chamber; a PCR reaction chamber; a waste liquid chamber; a capillary valve; 10: a gas passage.

[0221] More preferably, the structure of the bottom encapsulation sheet includes: a microfluidic chip mounting hole; a microfluidic chip encapsulation positioning hole; an RNaseA loading chamber; a ProteinaseK loading chamber.

[0222] The present invention also discloses a method for automatically enriching and / or detecting mutant genes and / or methylated DNA, comprising the following steps: 1) Sample loading: Load the pre-amplification system into the pre-amplification reaction chamber; add RNase A to the RNase A sample loading chamber; add Proteinase K to the Proteinase K sample loading chamber; add the qPCR system to the PCR reaction chamber; 2) Sealing; 3) Loading and completing the experiment: Load the sealed microfluidic chip onto the centrifugal microfluidic platform.

[0223] Compared with the prior art, the beneficial effects of the present invention are as follows: Based on the CRISPR system, programmable nucleases, and nucleic acid amplification technology, the present invention specifically cleaves wild-type nucleic acids using programmable nucleases while amplifying nucleic acids, enabling the number of mutant alleles in the sample to continuously increase exponentially to a level detectable by inexpensive Sanger sequencing. It not only achieves detection sensitivity comparable to ultra-deep NGS sequencing but also has characteristics such as a short detection cycle and no dependence on large-scale instrument equipment, greatly reducing the detection cost and significantly promoting 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. Description of the Drawings

[0224] Figure 1 It is the schematic diagram of enriching low-abundance mutant alleles in the present invention; Figure 2 It is the verification result diagram of sgRNA cleavage in Example 1; Figure 3 It is the sequencing results of wild-type alleles and ED2 before and after treating ED2 with SpCas9 in Example 1; Figure 4 It is the sequencing result of ED2 after treating ED2 with Hypa Cas9 in Example 1; Figure 5 It is the sequencing results of wild-type alleles and ED1 before and after treating ED1 with SpCas9 HF1 in Example 1; Figure 6 It is the sequencing result of ED1 after treating ED1 with Evo Cas9 in Example 1; Figure 7 It is the verification result diagram of sgRNA cleavage in Example 2; Figure 8 It is the sequencing results of wild-type alleles and mutant alleles before and after treating with HiFi Cas9 in Example 2; Figure 9 It is the sequencing result of mutant alleles after treating with SuperFi Cas9 in Example 2; Figure 10Sequencing results of mutant alleles processed with HiFi SC++ for 0 min in Example 2; Figure 11 Sequencing results of mutant alleles processed with HiFi SC++ for 5 min in Example 2; Figure 12 Sequencing results of mutant alleles processed with HiFi SC++ for 10 min in Example 2; Figure 13 Sequencing results of mutant alleles processed with HiFi SC++ for 15 min in Example 2; Figure 14 Sequencing results of mutant alleles processed with HiFi SC++ for 20 min in Example 2; Figure 15 Verification result diagram of sgRNA cleavage in Example 3; Figure 16 Sequencing results of wild-type and mutant alleles before and after treatment with Cas9 in Example 3; Figure 17 Comparison of enrichment levels of 0.1% methylated DNA before and after enrichment treatment in Example 4; Figure 18 Schematic diagram of the principle of enriching methylated DNA in the present invention; Figure 19 Sequencing results of wild-type and mutant alleles before and after treatment with Cas12 on NRAS in Example 5; Figure 20 Schematic diagram of the microfluidic chip structure; Figure 21 Schematic diagram of the top encapsulation sheet structure; Figure 22 Schematic diagram of the middle reaction layer structure; Figure 23 Schematic diagram of the top encapsulation sheet structure; Reference numerals in the drawings: 1 - top encapsulation sheet; 11 - microfluidic chip mounting hole; 12 - microfluidic chip encapsulation positioning hole; 13 - microfluidic chip injection hole; 2 - middle reaction layer; 21 - microfluidic chip encapsulation positioning hole; 22 - microfluidic chip mounting hole; 23 - pre-amplification reaction chamber; 24 - siphon valve; 25 - digestion reaction chamber; 26 - pre-distribution chamber; 27 - PCR reaction chamber; 28 - waste liquid chamber; 29 - capillary valve; 210 - gas passage; 3 - bottom encapsulation sheet; 31 - Microfluidic chip mounting hole; 32 - Microfluidic chip encapsulation positioning hole; 33 - RNaseA sample addition chamber; 34 - ProteinaseK sample addition chamber. Detailed implementation manners

[0225] Here, the exemplary embodiments will be described in detail. The implementation manners described in the following exemplary embodiments do not represent all implementation manners consistent with the present disclosure. On the contrary, they are merely examples of methods consistent with some aspects of the present disclosure.

[0226] In the following embodiments, the experimental methods, unless otherwise specified, are all conventional methods or are carried out according to the conditions recommended by the manufacturer. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels.

[0227] The primers used for sequencing in the following embodiments are the same as the qPCR primers.

[0228] Example 2 Enrichment and detection of BRAF gene V600E mutation 1. Design primers The human BRAF gene sequence was obtained from the NCBI database, and primers were designed based on this sequence as follows: Pre - amplification F primer: CTACACCTCAGATATATTTC (SEQ ID NO.19); Pre - amplification R primer: TGGATCCAGACAACTGT (SEQ ID NO.20); qPCR F primer: CCTCAGATATATTTCTTCATGA (SEQ ID NO.21); qPCR R primer: TGTTCAAACTGATGGGAC (SEQ ID NO.22).

[0229] 2. Design sgRNA sgRNA sequences with a length of 17 - 20 nt and capable of completely pairing with the wild - type allele sequence were designed, as shown in SEQ ID NO.2 - 6: guide1: UAGCUACAGUGAACUCUCGA (SEQ ID NO.2); guide2: UAGCUACAGUGAAAUCACGA (SEQ ID NO.3); guide3: GCUACAGUGAACUCUCGA (SEQ ID NO.4); guide4: GUCUAGCUACAGUGAAA (SEQ ID NO.5); guide5: GUCUAGCUGCAGUGAAA (SEQ ID NO.6).

[0230] 3. Genomic DNA extraction BRAF V600E mutant and wild-type alleles were respectively derived from B-CPAP and HCC827 cell lines (purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.); genomic DNA of the two cell lines was extracted using the QIAGEN DNeasy Blood & Tissue Kit according to the instructions, and the two DNAs were formulated into templates with mutant ratios of MAF = 5%, 1%, 0.5%, 0.1%, and 0.01% for mutant enrichment and detection.

[0231] 4. Cleavage verification of sgRNA a) Formation of ribonucleoprotein complex Prepare the following reaction system as shown in Table 6.

[0232] Table 6 Reaction system Final concentration Cas9 2.5 μM sgRNA 2.5 μM HEPES 1M Here, Cas9 includes one of HiFi Cas9, SuperFi Cas9, and HiFi SC++; Incubate at 37 °C for 10 min to obtain the ribonucleoprotein complex; b) Preparation of cleavage system, see Table 7.

[0233] Table 7 Cleavage system Final concentration Ribonucleoprotein complex 2.5 μM MgOAc 14 mM Template 0.25 μM Cut at 37 °C for 1 h; c) Digestion Add 1 μl of RNaseA (10 mg / ml), and place at room temperature for 10 min; add 1 μl of ProteinaseK (20 mg / ml), incubate at 56 °C for 30 min; then incubate at 95 °C for 10 min; d) Gel electrophoresis Use PAGE gel electrophoresis, add loading buffer to the cleavage product, and load the sample; electrophorese at 150 V for 10 min; After electrophoresis, stain with gelred dye for 15 min and then take a photo (see Figure 7), where lanes 1, 3, 5, 7, 9, and 11 contain wild-type nucleic acids, and lanes 2, 4, 6, 8, 10, and 12 contain mutant nucleic acids; by comparing lanes 1 and 2, 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12 respectively, it can be seen that wild-type alleles are more easily cleaved than mutant alleles; by comparing lanes 1, 3, 5, 7, 9, and 11, it is known that guide3 hardly cleaves wild-type alleles.

[0234] 5. Enrichment and detection of mutant alleles a) Pre-amplification: The formation of ribonucleoprotein complexes is shown in Table 8.

[0235] Table 8 Ribonucleoprotein complexes Final concentration Cas9 1 μM sgRNA 1 μM HEPES 1M Incubate at a constant temperature of 37 °C for 10 min to obtain ribonucleoprotein complexes; Prepare the pre-amplification system as shown in Table 9.

[0236] Table 9 Pre-amplification system Final concentration Ribonucleoprotein complex 1 μM Pre-amplification F primer 0.5 μM Pre-amplification R primer 0.5 μM ​ ​ ​ ​ ​ ​ ​ ​ ​ Here, Cas9 includes one of HiFi Cas9, SuperFi Cas9, and HiFi SC++; during specific operations, the Cas protein used to prepare ribonucleoprotein complexes in this step corresponds to the Cas9 used in step 4 for the cleavage verification of sgRNA; React at a constant temperature of 37 °C for 20 min; then terminate the reaction at 95 °C for 10 min; Digestion: Add 1 μl of RNaseA (10 mg / ml) and incubate at room temperature for 10 min; add 1 μl of Proteinase K (20 mg / ml) and incubate at a constant temperature of 56 °C for 30 min; then incubate at 95 °C for 10 min; Prepare the qPCR system as shown in Table 10.

[0237] Table 10 qPCR system ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ Reaction program: Pre-denaturation: 95 °C for 5 min; 45 cycles; 95 °C for 10 s; 60 °C for 30 s; After amplification, perform Sanger sequencing on the amplification products. The sequencing results are as ​ shown. To avoid redundancy, only the enrichment results of samples with MAF = 0.1% are shown; from ​It can be seen that the wild-type allele increases significantly after enrichment; moreover, by comparing ​ it can be known that as the treatment time with Cas protein increases, the enrichment effect becomes significantly better. This indicates that the enrichment method of the present invention can effectively enrich the mutant allele.

[0238] Example 3 Enrichment and detection of EGFR gene T790M mutation 1. Design primers Obtain the human EGFR gene sequence from the NCBI database and design primers according to this sequence as follows: Pre-amplification F primer: TACGTGATGGCCAGCGTGGA (SEQ ID NO.23); Pre-amplification R primer: ACTGGGAGCCAATATTGT (SEQ ID NO.24); qPCR F primer: TGATGGCCAGCGTGGACAA (SEQ ID NO.25); qPCR R primer: TTGTGTTCCCGGACATAGTC (SEQ ID NO.26).

[0239] 2. Design sgRNA Design sgRNA sequences with a length of 17 - 20 nt and capable of completely pairing with the wild-type allele sequence, as shown in SEQ ID NO.27 - 28: guide1: GGCAGCCGAAGGGCAUGAGC (SEQ ID NO.27); guide2: GGCAGCCGAAGAGCAUGAGC (SEQ ID NO.28); 3. Obtain the target gene The following are the short-chain sequences of the genes used as markers in this example: T790M-WT-S: GACATAGTCCAGGAGGCAGCCGAAGGGCATGAGCTGCGTGATGAGCTGCACGGTGGAGGTGAGGCAGATGCCCAGCAGGC (SEQ ID NO.29); T790M-WT-AS: GCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATCACGCAGCTCATGCCCTTCGGCTGCCTCCTGGACTATGTC (SEQ ID NO.30); T790M-MUT-S: GACATAGTCCAGGAGGCAGCCGAAGGGCATGAGCTGCATGATGAGCTGCACGGTGGAGGTGAGGCAGATGCCCAGCAGGC (SEQ ID NO.31); T790M-MUT-AS: GCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATCATGCAGCTCATGCCCTTCGGCTGCCTCCTGGACTATGTC (SEQ ID NO.32); Synthesized by Shanghai Sangon Biotech Co., Ltd.

[0240] Obtaining genomic DNA: The wild-type allele of EGFR T790M was from the B-CPAP cell line, purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd., and genomic DNA was extracted using the QIAGEN DNeasy Blood & Tissue Kit. The standard product of the EGFR T790M mutant allele sequence was purchased from GeneCopoeia. Genomic DNA and the standard product of the mutant allele sequence were formulated into templates with mutant ratios MAF = 5%, 1%, 0.5%, 0.1%, and 0.01%.

[0241] 4. Cleavage verification of sgRNA a) Formation of ribonucleoprotein complex, see Table 11.

[0242] Table 11 Ribonucleoprotein complex ​ ​ ​ ​ ​ ​ 1M Incubate at a constant temperature of 37°C for 10 min; b) Preparation of cleavage system, see Table 12.

[0243] Table 12 Cleavage system ​ ​ ​ ​ ​ ​ ​ Cut at a constant temperature of 37°C for 1 h; c) Digestion Add 1 μl of RNaseA (10 mg / ml), place at room temperature for 10 min; add 1 μl of Proteinase K (20 mg / ml), incubate at a constant temperature of 56°C for 30 min; then incubate at 95°C for 10 min.

[0244] d) Gel electrophoresis Use PAGE gel electrophoresis, add loading buffer to the cut product, load the sample; perform electrophoresis at 150V for 10 min; After electrophoresis, stain with gelred dye for 15 min and then take a photo (see ​ ). ​ Among the 2 lanes from left to right are wild type and mutant type in

[0245] 5. Enrichment and detection of mutant alleles a) Pre-amplification: The formation of ribonucleoprotein complex is shown in Table 13.

[0246] Table 13 Ribonucleoprotein complex ​ ​ ​ ​ ​ ​ 1M Incubate at a constant temperature of 37°C for 10 min to obtain ribonucleoprotein complex; Prepare the pre-amplification system as shown in Table 14.

[0247] Table 14 Pre-amplification system ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ React at a constant temperature of 37°C for 20 min; then terminate the reaction at 95°C for 10 min; Digestion: Add 1 μl of RNaseA (10 mg / ml), place at room temperature for 10 min; add 1 μl of Proteinase K (20 mg / ml), incubate at 56°C for 30 min; then incubate at 95°C for 10 min; Prepare the qPCR system as shown in Table 15.

[0248] Table 15 qPCR system ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ Reaction program: Pre-denaturation: 95°C for 5 min; 45 cycles; 95°C for 10 s; 60°C for 30 s; After amplification, perform sanger sequencing on the amplified product. The sequencing results are as ​ shown. To avoid redundancy, only the enrichment results of samples with MAF = 5% are shown; as ​ can be seen, the wild-type alleles increase significantly after enrichment. It shows that the enrichment method of the present invention can effectively enrich mutant alleles.

[0249] Example 4 Enrichment and detection of PCDH10 gene methylation 1. Design primers The human PCDH10 gene sequence was obtained from the NCBI database, and primers were designed based on this sequence as follows: Pre-amplification F primer: TCGTTAAATAGATACGTTACGC (SEQ ID NO.33); Pre-amplification R primer: TAAAAACTAAAAACTTTCCGCG (SEQ ID NO.34); qPCR F primer: TCGTTAAATAGATACGTTACGC (SEQ ID NO.35); qPCR R primer: CAACGCCTCGAAACCTACG (SEQ ID NO.36).

[0250] 2. Design of sgRNA Design an sgRNA sequence with a length of 17 - 20 nt that can fully pair with the wild-type allele sequence, as shown in SEQ ID NO.37: guide: AAUUUUUGUUUGAGUGGUUG (SEQ ID NO.37); 3. Obtaining genomic DNA A549 is a PCDH10 non-methylated cell line, purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. Genomic DNA was extracted using the QIAGEN DNeasy Blood&Tissue Kit. Methylated Human Control was purchased from promega. Templates with methylation ratios of MAF = 5%, 1%, 0.5%, 0.1%, and 0.01% were prepared.

[0251] 4. Enrichment and detection of methylated DNA a) Pre-amplification: Formation of ribonucleoprotein complex, see Table 16.

[0252] Table 16 Composition of ribonucleoprotein complex ​ ​ ​ ​ ​ ​ 1M Incubate at 37°C for 10 min to obtain the ribonucleoprotein complex; Prepare the pre-amplification system, see Table 17.

[0253] Table 17 Pre-amplification system ​ ​ 1 μM Pre-amplification F primer 0.5 μM Pre-amplification R primer 0.5 μM MgOAc 14 mM dNTP 0.45 mM DNA polymerase Recombinase SSB Template 60 ng React at 37°C for 20 min; then terminate the reaction at 95°C for 10 min; Digestion: Add 1 μl RNase A (10 mg / ml) and let it stand at room temperature for 10 min; add 1 μl Proteinase K (20 mg / ml) and keep it at 56°C for 30 min; then keep it at 95°C for 10 min; For qPCR system preparation, see Table 18.

[0254] Table 18 qPCR system Final concentration qPCR F primer 1 μM qPCR R primer 1 μM MgOAc 14 mM dNTP 0.45 mM DNA polymerase Reaction procedure: Pre-denaturation: 95°C, 5 min; 30 cycles; 95℃, 10s; 60℃, 30s; 72℃, 30s.

[0255] After amplification, the amplified product was subjected to Sanger sequencing, and the sequencing results were as follows: Figure 17 To avoid redundancy, only the enrichment results of samples with MAF=0.1% are shown; Figure 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 methylated DNA.

[0256] Example 6 PASEA Automation The enrichment and detection experiments in the above examples can be automated using a microfluidic chip. The microfluidic chip structure (see Figure 20 ) is divided into three parts, consisting of a top encapsulation sheet 1, a bottom encapsulation sheet 3 and a middle reaction layer 2; Top package sheet 1 (see Figure 21 ) The structure is: microfluidic chip mounting hole 11; microfluidic chip packaging positioning hole 12; microfluidic chip injection hole 13; Intermediate reaction layer 2 (see Figure 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.

[0257] Bottom package sheet 3 (see Figure 23 ) structure is: microfluidic chip mounting hole 31; microfluidic chip packaging positioning hole 32; RNaseA sample addition cavity 33; ProteinaseK sample addition cavity 34.

[0258] The specific steps are as follows: 1) Sample loading: Load the pre-amplification system into the pre-amplification reaction chamber; Add 5 μl of RNase A (10 mg / ml) to the RNase A sample loading chamber; Add 5 μl of Proteinase K (20 mg / ml) to the Proteinase K sample loading chamber; Load the qPCR system into the PCR reaction chamber.

[0259] 2) Sealing: Seal the injection port with a suitable sealing aluminum foil pressure-sensitive membrane.

[0260] 3) Load and complete the experiment: Load the sealed microfluidic chip onto the centrifugal microfluidic platform.

[0261] Set the process: a: Incubate at 37 °C for 20 min (pre-amplification); b: Rotate at 3000 rpm for 1 min (centrifuge the pre-amplified solution to the digestion chamber during rotation and open the siphon valve when stopped); c: Leave at room temperature for 10 min (RNase A digestion); d: Incubate at 56 °C for 30 min (Proteinase K digestion); e: Rotate at 1000 rpm for 1 min (pre-distribution); f: Rotate at 3000 rpm for 2 min (centrifuge the quantified digested pre-amplified solution to the PCR chamber); g: 95 °C, 5 min; 45 cycles: 95 °C, 10 s 60 °C, 30 s (PCR reaction).

[0262] The routine operations in the operation steps of the present invention are well-known to those skilled in the art and will not be elaborated here.

[0263] The above-described embodiments have described the technical solutions of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the present invention. Any modifications, supplements, or substitutions in a similar manner within the principle scope of the present invention should be included within the protection scope of the present invention.

Claims

1. A kit for the enrichment of mutant genes based on programmable enzymes, comprising: A ribonucleoprotein complex, the ribonucleoprotein complex comprising component (a) and component (b): Component (a), the component (a) comprising: a nucleic acid region capable of complementary base pairing with a target nucleic acid and a 0-4 bp mismatch fragment; or a nucleic acid region capable of complementary base pairing with a target nucleic acid and an adjacent PAM comprising a mutation-prone site; Component (b), capable of binding to a target nucleic acid and cleaving the target nucleic acid strand; Wherein, the component (a) is selected from tracrRNA and / or crRNA and / or sgRNA and / or derivatives of the three and / or guide DNA and / or guide RNA; the component (b) comprises a Cas protein and / or a Cas protein derivative; the components (a) and (b) are capable of binding.

2. The kit according to claim 1, characterized in that The spacer sequence length of sgRNA, guide DNA or guideRNA in the component (a) is 16-22 nt.

3. The kit according to claim 1, characterized in that The component (b) comprises at least one of the following: Cas12a, SacCas9, CjCas9, SpCas9, NmCas9, Sp-Cas9 HF1, evoCas9, HypaCas9, HiFi Cas9, Sniper-Cas9, xCas9, eSpCas9 1.1, SuperFi Cas9, SaCas9, SaCas9-HF, efSaCas9, ScCas9, Cas9-Sc++, Cas9 HiFi-Sc++, SpaCas9, SpaCas9-HF, FnCas9, AnaCas9, SpyCas9, FnCas12a, LbCas12a or AsCas12a.

4. The kit according to claim 1, characterized in that, The mutant gene comprises a cancer-related gene.

5. The kit according to claim 4, wherein The cancer-related gene comprises 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.

6. The kit according to claim 1, characterized in that, The kit further comprises an isothermal cleavage-amplification reaction system, the isothermal cleavage-amplification reaction system comprising: primers, a target gene, an enzyme and a chromogenic group.

7. The kit according to claim 1, characterized in that, The sgRNA comprises the following sequences: SEQ ID NO.1: UCUUAAUUCCUUGAUAGCGA; SEQ ID NO.2: UAGCUACAGUGAACUCUCGA; SEQ ID NO.3: UAGCUACAGUGAAAUCACGA; SEQ ID NO.4: GCUACAGUGAACUCUCGA; SEQ ID NO.5: GUCUAGCUACAGUGAAA; SEQ ID NO.6: GUCUAGCUGCAGUGAAA; SEQ ID NO.27: GGCAGCCGAAGGGCAUGAGC; SEQ ID NO.28: GGCAGCCGAAGAGCAUGAGC; SEQ ID NO.37: AAUUUUUGUUUGAGUGGUUG; SEQ ID NO.42: UCCAGCUGUAUCCAGUAUGU.

8. The kit according to claim 1, wherein The enrichment includes automated enrichment; the detection includes automated detection, and the automated enrichment includes enrichment using a microfluidic chip; The automated detection includes detection using a microfluidic chip.

9. A method for enriching mutant genes using the kit according to claim 1, comprising the following steps: Collecting a sample; Preparing a ribonucleoprotein complex; Formulating a reaction system; Enrichment analysis.

10. Use of the Cas protein, including at least one of the following: (1) Enriching gene mutations; (2) Detecting gene mutations; (3) Enriching methylated DNA; or, (4) Detecting methylated DNA; Among them, 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.

Images