CN110117818A - Detect the CRISPR high flux biochip of single gene mutation - Google Patents

Abstract

The invention discloses a CRISPR high-throughput biochip for detecting single-gene mutation, which utilizes the non-specificity of single-stranded DNA cleavage produced by CRISPR-Cas12a protein after specific cleavage of double-stranded DNA, and is detected by Cas12a through a single-stranded fluorescent probe. The fluorescence intensity changes after the non-specific cleavage of the protein is used to construct a CRISPR high-throughput biochip for detecting single gene mutation. The invention uses hydrophobin to modify the chip substrate. Compared with ordinary substrates, the hydrophobin-modified chip substrate not only makes the hydrophilic protein Cas12a spread evenly on the chip surface, but also avoids the waste of protein and crRNA, and ensures that the target gene is at a low level. Compared with ordinary single-gene disease detection, it has the characteristics of high efficiency, low cost, short cycle and strong specificity, and the detection material is stable , The operation difficulty is low, the results are intuitive and reliable, and it is very expected to be put into practical application.

Description

CRISPR high-throughput biochips for detection of single-gene mutations

technical field

The invention belongs to the field of biotechnology, in particular, the invention relates to a CRISPR high-throughput biochip for detecting single gene mutation and a construction method.

Background technique

Monogenic diseases mainly refer to diseases caused by a pair of allele mutations, which are divided into two cases caused by dominant gene mutations and recessive gene mutations. In clinical diagnosis, there are many diseases caused by single gene mutation. Since monogenic diseases have unique characteristic gene mutation sites, nucleic acid molecular tests for specific monogenic diseases can be developed. Nucleic acid diagnosis (NADs nucleic acid diagnostics) is to use the theory and technology of molecular biology to analyze the function of nucleic acid from the nucleic acid structure, replication, transcription or translation process by directly probing the existence state of specific nucleic acid in different species, so as to provide targeted diagnosis and treatment. method of making a diagnosis. Similar to this is the detection of single nucleotide polymorphisms (SNPs, single nucleotide polymorphisms). SNPs are widely present in the human genome, and their frequency is about 1% or higher. The development of SNPs detection technology is very important for genetic samples. Detection is especially important.

However, the above-mentioned single-gene mutation disease detection not only has the technical problems of non-human operation, but also can only detect a few or even one single-gene disease at a time. In addition, the detection technology costs are too high. These technologies are used in the detection of single-gene mutation diseases. is not widely used.

Due to the high stability of DNA molecules, most of the current NADs methods detect DNA molecules, and some methods detect RNA molecules. The steps of nucleic acid molecule detection are roughly two steps: the first step is the amplification of the target DNA, and the second step is the detection of the target DNA. Nucleotide changes in SNPs come in two forms: transitions and transversions. With the continuous development of biotechnology, a class of high-throughput and highly automated SNP detection methods have emerged, such as direct sequencing and DNA chips. At present, the American company Aofei has made high-throughput biochips for SNPs detection by using molecular hybridization and in situ fluorescence detection, but the cost of chip design is high, and some SNPs cannot be detected.

The biochip integrates the biochemical analysis process on the surface of the chip based on the principle of specific interaction between biomolecules, so as to achieve high-throughput and rapid detection of DNA, RNA, peptides, proteins and other biological components. It can be used as an operating platform for various detection methods, that is, it can simultaneously perform experimental analysis of a large number of different reaction conditions and output data, so as to achieve simultaneous and high-speed detection of different targets.

Hydrophobin is a kind of small molecular weight (about 12KD) protein with special physical and chemical properties produced by higher filamentous fungi in a specific period. Its protein structure is relatively special, containing both hydrophilic and hydrophobic structures. for biparental. The amphiphilic nature of hydrophobins makes them like surfactants whose main function is to self-assemble on any hydrophilic/hydrophobic surface to form an amphiphilic membrane to change the surface properties. Hydrophobin is one of the known proteins with the highest surface activity. It has the characteristics of high temperature resistance, acid and alkali resistance, etc. It can be used to adjust the structure of protein molecular arrangement in actual operation to achieve higher reaction efficiency. High theoretical value and application value.

The CRISPR-Cas12a protein is an RNA-guided enzyme as a component of the bacterial adaptive immune system that possesses a T-rich protospacer-adjacent motif (PAM) that catalyzes the maturation of their own guide RNAs (crRNAs). And generate an irregular double-stranded DNA breakpoint at the end of the PAM. After specific cleavage, Cas12a protein can also indiscriminately cut DNA single strands non-specifically. Using this unique property, we can detect whether the system completes the cleavage function through fluorescent probes.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a CRISPR high-throughput biochip for detecting single gene mutation, which overcomes the deficiencies of the prior art biochips such as low throughput, high cost, technical defects and the like.

The technical scheme of the present invention is:

A CRISPR high-throughput biochip for detecting single gene mutation, the construction method includes the following steps:

(1) Chemical synthesis of an E. coli expression vector containing the Cas12a protein sequence, and a specific substrate containing PAM (PAM refers to the adjacent motif of the protospacer sequence, which is a necessary recognition sequence for Cas12a to perform the cleavage function, and the PAM sequence of FnCas12a is TTN). primers, PCR upstream primers containing PAM sequences, downstream primers, fluorescent probes, and crRNAs corresponding to FnCas12a and corresponding single-gene mutation diseases (crRNA, namely guide RNA, refers to the RNA that guides FnCas12a protein to specifically bind to target DNA, crRNA about 43bp), determine the expression of Cas12a protein in E. coli recombinant plasmid;

(2) The complex of Cas12a and crRNA to specific substrates (including synthetic plasmids and fragments of about 140 bp in length obtained by PCR amplification by designing upstream primers containing PAM) - a system for specific cleavage of double-stranded DNA Construction, including optimization of the ratio of double-stranded DNA cutting experiments, and cutting of PCR amplified fragments;

(3) Construction of a system for non-specific cleavage of single-stranded DNA (including synthetic single-stranded DNA and fluorescent probes) by the complex of Cas12a and crRNA, including the cleavage of common single-stranded DNA, the cleavage of fluorescent probe single-stranded DNA cutting;

(4) modifying the chip substrate with hydrophobin;

(5) Construction of a high-throughput chip substrate, including the immobilization of FnCas12a-crRNA complexes by hydrophobin, the specific cleavage of specific substrates by the complexes, and the non-specific cleavage of fluorescent probes.

In the step (4), hydrophobin refers to some amphiphilic proteins that can form a nanometer-thick amphiphilic protein membrane through self-assembly at the two-phase interface. Said step (4) hydrophobin refers to HGFI and HFBI.

The Cas12a protein is preferably FnCas12a protein.

The construction method of CRISPR high-throughput biochip for detecting single gene mutation includes the following steps:

(1) Chemically synthesize E. coli expression vector containing Cas12a protein sequence, specific substrate, PCR upstream primer, downstream primer, fluorescent probe containing PAM sequence, and crRNA corresponding to Cas12a and corresponding single gene mutation disease, determine FnCas12a Protein expression in E. coli recombinant plasmids;

(2) Construction of a system for the specific cleavage of a specific substrate, double-stranded DNA, by the complex of Cas12a and crRNA, including the optimization of the experimental ratio of double-stranded DNA cleavage and the cleavage of PCR-amplified fragments;

(3) Construction of a system for non-specific cleavage of single-stranded DNA by the complex of Cas12a and crRNA, including the cleavage of common single-stranded DNA and the cleavage of fluorescent probe single-stranded DNA;

(4) modifying the chip substrate with hydrophobin;

(5) Construction of a high-throughput chip substrate, including the immobilization of the Cas12a-crRNA complex by hydrophobin, the specific cleavage of the specific substrate and the non-specific cleavage of the fluorescent probe by the complex.

In the step (4), hydrophobin refers to some amphiphilic proteins that can form a nanometer-thick amphiphilic protein membrane through self-assembly at the two-phase interface. Said step (4) hydrophobin refers to HGFI and HFBI.

The Cas12a protein is preferably FnCas12a protein.

Application of CRISPR high-throughput biochips for detection of single-gene mutations.

Beneficial effects of the present invention: The present invention uses hydrophobin to modify the chip substrate. Compared with ordinary substrates, the chip substrate modified by hydrophobin not only makes the hydrophilic protein Cas12a spread evenly on the chip surface, but also avoids the waste of protein and crRNA. It ensures that the target gene still has good detection results at low concentrations, and realizes the detection of 1 copy of the target gene. Compared with ordinary single-gene disease detection, it has the characteristics of high efficiency, low cost, short cycle and strong specificity , and the detection material is stable, the operation difficulty is low, the results are intuitive and reliable, and it is very expected to be put into practical application.

Description of drawings

Figure 1 shows the Western Blots of two different sources of Cas12a—FnCas12a and LbCas12a;

Figure 2 shows an agarose gel electrophoresis profile of a specific substrate; in which:

Figure 2A shows the agarose gel electrophoresis images of two plasmids pDsRed-Monomer-N1 and pAcGFP1-N1;

Figure 2B shows the agarose gel electrophoresis image of the transcription activator GATA4 expression vector;

Figure 2C shows an agarose gel electrophoresis image of the PCR fragment of plasmid pAcGFP1-N1;

Figure 3 shows the properties of FnCas12a cleavage-specific double-stranded DNA; where:

Fig. 3A is the result of protein gradient electrophoresis of FnCas12a specific cleavage plasmid;

Fig. 3B is the plasmid gradient electrophoresis result of FnCas12a specific cutting plasmid;

Figure 3C is a graph showing the results of optimized sample ratio gradient electrophoresis of FnCas12a specific cleavage plasmid;

Figure 3D is the electrophoresis result of the optimal sample ratio of the plasmid specifically cut by FnCas12a;

Fig. 3E is the electrophoresis result of the optimal sample ratio of the two plasmids specifically cut by FnCas12a;

Fig. 3F is the electrophoresis result figure of the PCR fragment of FnCas12a specific cleavage plasmid;

Figure 4 shows the properties of FnCas12a for cleavage of non-specific single-stranded DNA; where:

Figure 4A shows the cleavage of non-specific single-stranded DNA-EGFP by FnCas12a;

Figure 4B shows the result of single-stranded DNA gradient electrophoresis of FnCas12a cleaving non-specific single-stranded DNA;

Figure 5 shows the fluorescence values of FnCas12a non-specifically cleaved fluorescent probes; where:

Figure 5A shows the probe gradient fluorescence value of FnCas12a non-specifically cleaved fluorescent probe;

Figure 5B shows the probe minimum detection line fluorescence value of FnCas12a cleavage fluorescent probe;

Figure 5C shows that the material of FnCas12a non-specifically cleaved the fluorescent probe affects the fluorescence value;

Figure 6 shows a schematic flow chart of the CRISPR cleavage reaction of the hydrophobin modified chip;

Figure 7 shows the results of fluorescence detection of on-chip CRISPR cleavage reactions with and without hydrophobin modification.

Detailed ways

The technical solutions in the present invention will be further described below with reference to the accompanying drawings in the specific embodiments of the present invention.

the term:

The term "guide RNA" refers to an RNA that directs a Cas protein to specifically bind to a target DNA sequence.

The term "crRNA" refers to CRISPR RNA, which is a short RNA that guides Cas12a to bind to a target DNA sequence.

The term "CRISPR" refers to the clustered regularly interspaced short palindromic repeats, which are the immune system of many prokaryotes.

The term "Cas protein" refers to a CRISPR-associated protein, which is an associated protein in the CRISPR system.

The term "Cas12a" (formerly "Cpf1") refers to the crRNA-dependent endonuclease, which is a type V enzyme in the CRISPR system classification.

The term "PAM" refers to the protospacer-adjacent motif, which is necessary for Cas12a cleavage. The PAM of FnCas12a is the TTN sequence, and the PAM of LbCas12a is the TTTN sequence.

Material:

RNase inhibitors were purchased from Solarbio; primers (oligonucleotides), single-stranded DNA fragment DNMT, guide RNAs including pAcGFP1-N1-1, pDsRed-Monomer-N1-1, DNMT-1, FnCas12a protein expression vector, LbCas12a The protein expression vectors were purchased from GENEWIZ Company; the fluorescent probe (FAM-TTATT-BHQ1) was purchased from Beijing Saibaisheng Gene Technology Co., Ltd.;

pAcGFP1-N1 and pDsRed-Monomer-N1 are commercially available.

Escherichia coli (E.coli) DH5α and BL21 are commercially available.

Example 1: Gradient experiment of optimized sample ratio for specific cleavage of pAcGFP1-N1 plasmid by FnCas12a

1. Transformation of E.coli BL21 of FnCas12a protein expression vector and purification of FnCas12a protein, the specific steps are as follows:

The transformation of E.coli BL21 of the FnCas12a protein expression vector was carried out as follows:

1. Take a tube of competent cells E.coli BL21 and dissolve slowly on ice, add the FnCas12a protein expression vector plasmid to be transformed (10μL), mix gently and then ice bath for 30min;

2. After heat shock at 42°C for 90s, quickly ice bath for 5min;

3. Add 900 μL of LB medium with a warm bath at 37°C, and incubate at 37°C for 1 hour;

4. Centrifuge at 3000 rpm for 3 min, in a sterile operating table, discard 500 ul of the bacterial solution with a pipette and mix gently, take 200 μl and spread it on the LB selection plate containing kanamycin (100 μg/mL), 37 Invert at ℃ for 12-16h;

5. Pick a single clone for subsequent plasmid extraction and verification experiments.

In the above steps, the positive clone verification of the plasmid vector is carried out as follows:

1. Pick the transformant of the FnCas12a protein expression vector from the plate, insert it into 5mL LB liquid medium (containing 100μg/mL kanamycin), and cultivate it on a shaker at 37°C for 12-16h;

2. Take 5mL of bacterial liquid and extract plasmid with plasmid mini-extraction kit;

3. Take 1 μL of the plasmid extract for Nanodrop detection to detect the concentration and purity of the extracted plasmid;

4. Store the extracted plasmid at -20°C.

In the above steps, FnCas12a protein purification was carried out as follows:

1. Pick an appropriate amount of single clones with obvious surrounding colonies on the plate, insert them into 5mL LB liquid medium (containing 100μg/mL kanamycin), and cultivate at 37℃ for 6h on a shaker;

2. Put the bacterial liquid with an OD value of about 0.5 into a 1L LB liquid culture bottle (containing 100 μg/mL kanamycin), and cultivate at 37°C for 6 hours on a shaker;

3. Adjust the temperature of the shaker to 16°C, cool down for 30 minutes, add 500 μl IPTG to each bottle of medium for induction, and culture at 16°C on a shaker for 16 hours;

4. Centrifuge the bacteria at 4000rpm and 4°C for 15min, resuspend the bacteria with washing buffer [50mM Tris-HCl (pH8.0), 1.5MNaCl, 5%glycerol], and break the bacteria with a sterilizer;

5. Centrifuge at 18000 rpm for 40 min at 4 °C, collect the supernatant, perform affinity chromatography, and run the gel for verification; change the medium with 50 mM Tris-HCl (pH 8.0), 1 mM DTT, 5% glycerol, and dilute to a salt concentration of 80 mM the following;

6. Purify by protein chromatography and run the gel by SDS-PAGE electrophoresis. Figure 1 shows the Western Blot of FnCas12a and LbCas12a.

2. Transformation of Escherichia coli (E.coli) DH5α of pAcGFP1-N1 and pDsRed-Monomer-N1 plasmids, the specific steps are as follows:

1. Take 2 tubes of competent cells E.coli DH5α and dissolve slowly on ice, add the plasmids to be transformed pAcGFP1-N1 and pDsRed-Monomer-N1 (10 μL each), mix gently and then ice bath for 30 min;

2. After heat shock at 42°C for 90s, quickly ice bath for 5min;

3. Add 900 μL of LB medium with a warm bath at 37°C, and incubate at 37°C for 1 hour;

4. Centrifuge at 3000 rpm for 3 min. In a sterile operating table, discard 500 ul of the bacterial solution with a pipette and mix gently. Take 200 μl of each and apply them to LB selection plates containing kanamycin (100 μg/mL). Invert at 37°C for 12-16h;

5. Pick a single clone for subsequent plasmid extraction and verification experiments.

In the above steps, the positive clone verification of the plasmid vector is carried out as follows:

1. Pick the transformants of pAcGFP1-N1 and pDsRed-Monomer-N1 from the plate respectively, insert them into 5mL LB liquid medium (containing 100μg/mL kanamycin), and culture at 37°C on a shaker for 12-16h;

2. Take 5 mL of bacterial solution and extract plasmids with a plasmid mini-extraction kit;

3. Take 1 μL of the plasmid extracts for Nanodrop detection to detect the concentration and purity of the extracted plasmids;

4. Store the extracted plasmid at -20°C. Figure 2A shows agarose gel electrophoresis images of two plasmids, pDsRed-Monomer-N1, pAcGFP1-N1.

3. The protein gradient optimization experiment of specific cleavage of pAcGFP1-N1 plasmid by FnCas12a, the specific steps are as follows:

1. In four 20uL reaction systems, add FnCas12a (62.5nM, 125nM, 187.5nM, 250nM) purified in step 1, guide RNA-pAcGFP1-N1-1 (0.5uM), pAcGFP1-N1 plasmid 1uL, RNase Inhibitor 0.5uL, buffer NEB buffer 3 to make up.

2. The reaction was carried out at 37°C for 15 minutes, and then terminated at 98°C for 2 minutes.

3. Perform polyacrylamide gel electrophoresis. Figure 3A shows the result of protein gradient electrophoresis of the plasmid specifically cleaved by FnCas12a. The results showed that whether the specific substrate could be completely cleaved was related to the content of FnCas12a protein.

4. The plasmid gradient optimization experiment for the specific cleavage of pAcGFP1-N1 by FnCas12a, the specific steps are as follows:

1. To the six 20uL reaction systems, add FnCas12a (125nM) purified in step 1, respectively.

RNA——pAcGFP1-N1-1 (0.5uM), pAcGFP1-N1 plasmid (250ng, 300ng, 350ng, 400ng, 450ng, 500ng), RNase inhibitor 0.5uL, buffer NEB buffer 3 to fill.

2. The reaction was carried out at 37°C for 15 minutes, and then terminated at 98°C for 2 minutes.

3. Perform polyacrylamide gel electrophoresis. Figure 3B shows the result of plasmid gradient electrophoresis of the plasmid specifically cleaved by FnCas12a. The results showed that the amount of specific substrate and the amount of material should have a certain appropriate ratio.

5. The sample ratio gradient optimization experiment of FnCas12a specifically cutting pAcGFP1-N1 plasmid, the specific steps are as follows:

1. In three groups of 20uL reaction systems, respectively add FnCas12a (0.5uM, 1.25uM, 2.5uM, 5uM, 12.5uM, 25uM) purified in step 1, wherein the first group of guide RNAs—pAcGFP1-N1-1 (500nM ), the second group of guide RNAs—pAcGFP1-N1-1 (250nM), the third group of guide RNAs—pAcGFP1-N1-1 (50nM), pAcGFP1-N1 plasmid (1uL), RNase inhibitor 0.5uL, buffer Make up with NEB buffer 3.

2. The reaction was carried out at 37°C for 15 minutes, and then terminated at 98°C for 2 minutes.

3. Perform polyacrylamide gel electrophoresis, and FIG. 3C shows the results of the sample ratio gradient optimization of the plasmid specifically cleaved by FnCas12a. The results indicated that the ratio of guide RNA to substrate was decisive for the specific substrate cleavage result. 6. The optimal sample ratio experiment of FnCas12a specifically cutting pAcGFP1-N1 plasmid, the specific steps are as follows:

1. In the 20uL reaction system, add FnCas12a (0.25uM) purified in step 1.

RNA——pAcGFP1-N1-1 (0.25uM), pAcGFP1-N1 plasmid (1uL), RNase inhibitor 0.5uL, buffer NEB buffer 3 to make up.

2. The reaction was carried out at 37°C for 15 minutes, and then terminated at 98°C for 2 minutes.

3. Perform polyacrylamide gel electrophoresis. Figures 3D and 3E show the results of the optimal ratio electrophoresis of the samples (10:10:1) of the specific cleavage of pAcGFP1-N1 and pDsRed-Monomer-N1 plasmids by FnCas12a.

Example 2: Experimental design and construction of PCR fragments of FnCas12a specifically cutting pAcGFP1-N1 and pDsRed-Monomer-N1 plasmids, including the following steps:

1. Same as step 1 in Example 1.

2. Same as step 2 of Example 1.

3. Using PCR to obtain pAcGFP1-N1 and pDsRed-Monomer-N1 plasmid fragments, the specific steps are as follows:

The upstream primers GFP-F/RED-F and the downstream primers GFP-R/RED-R were designed, and the purified pAcGFP1-N1 and pDsRed-Monomer-N1 plasmids were used as templates to amplify the pAcGFP1-N1 and pDsRed-Monomer-N1 plasmid fragments. After the PCR was completed, it was directly used in the FnCas12a cleavage reaction. Figure 2C shows an agarose gel electrophoresis image of the PCR fragment of plasmid pAcGFP1-N1;

GFP-F: GTGGCGATAAGTCGTGTCTTACCGGGT

GFP-R: GTCAAACCGCTATCCACGCCCATTGATG

RED-F: GTACAAGGCCAAGAAGCCCGTGCAG

RED-R: GTCTCTTGATCGATCTTTGCAAAAGCCTAGGC

4. FnCas12a specifically cuts pAcGFP1-N1 and pDsRed-Monomer-N1 plasmid fragments. The specific steps are as follows:

1. In the two 30uL reaction systems, respectively add purified FnCas12a (250nM), guide RNAs—pAcGFP1-N1-1 (250nM), pDsRed-Monomer-N1-1 (250nM), pAcGFP1-N1 plasmid fragment (10uL) , pDsRed-Monomer-N1 plasmid fragment (10uL), RNase inhibitor 0.5uL, buffer NEB buffer3 complement.

2. The reaction was carried out at 37°C for 15 minutes, and then terminated at 98°C for 2 minutes.

3. Perform polyacrylamide gel electrophoresis. Figure 3F shows the electrophoresis result of the specific cleavage of pAcGFP1-N1 and pDsRed-Monomer-N1 plasmid fragments by FnCas12a. The results indicated that FnCas12a could specifically cut DNA fragments.

Example 3: Experimental design and construction of short-sequence electrophoresis of non-specific cleavage of single-stranded DNA by FnCas12a—DNMT, including the following steps:

1. Same as step 1 in Example 1.

2. Same as step 2 of Example 1.

3. Verification of the nature of non-specific cleavage of single-stranded DNA by FnCas12a, the specific steps are as follows:

1. In a 30uL reaction system, add purified FnCas12a (1uM), guide RNAs—pAcGFP1-N1-1 (0.5uM), DNMT-1 (0.5uM), pAcGFP1-N1 plasmid (1uL) as shown in the table in Figure 4A , DNMT DNA fragment (1uL), single-stranded DNA-EGFP (10uM), RNase inhibitor 0.5uL, buffer NEB buffer 3 to complete.

2. The reaction was carried out at 37°C for 15 minutes, and then terminated at 98°C for 2 minutes.

3. Prepare a non-denaturing polyacrylamide gel with a concentration of 12%, and perform polyacrylamide gel electrophoresis. Figure 4A shows the result of short-sequence non-denaturing electrophoresis of FnCas12a non-specifically cleaved single-stranded DNA-DNMT. The results showed that FnCas12a could only non-specifically cut single-stranded DNA, but not double-stranded DNA.

4. Experimental optimization of non-specific cleavage of single-stranded DNA by FnCas12a, the specific steps are as follows:

1. In the four 30uL reaction systems, purified FnCas12a (0.5uM), guide RNA-DNMT-1 (0.5uM), DNMT DNA fragment (0.5uM), single-stranded DNA- -EGFP (1uM, 2uM, 5uM, 6.25uM), RNase inhibitor 0.5uL, buffer NEB buffer 3 to make up.

2. The reaction was carried out at 37°C for 15 minutes, and then terminated at 98°C for 2 minutes.

3. Configure a denaturing polyacrylamide gel with a concentration of 20%, and perform polyacrylamide gel electrophoresis. Figure 4B shows the result of short-sequence denaturation electrophoresis of FnCas12a non-specifically cleaved single-stranded DNA-DNMT. The results indicated that the amount of non-specific cleavage of single-stranded DNA by FnCas12a was related to the amount of FnCas12a, guide RNA and specific substrate.

Example 4: Experimental design and construction of fluorescence detection of FnCas12a non-specifically cleaved fluorescent probe, including the following steps:

1. Same as step 1 in Example 1.

2. Same as step 2 of Example 1.

3. The construction of a probe gradient fluorescence numerical detection experiment for the non-specific cleavage of fluorescent probes by FnCas12a, including the following steps:

1. In 8 groups of 100uL reaction systems, purified FnCas12a (250nM) was added to the reaction system.

RNA - pAcGFP1-N1-1 (250nM), pAcGFP1-N1 plasmid (4uL), fluorescent probe (its constituent sequence is TTATT, and the 5' end is labeled with a fluorophore FAM, and the 3' end is labeled with a quencher group BHQ1, namely FAM-TTATT-BHQ1) (0.5uM, 1uM, 1.5uM, 2uM, 2.5uM, 3uM, 3.5uM, 4uM), RNase inhibitor 2uL, buffer NEB buffer 3 to complete, each group was repeated three times .

2. The reaction was carried out at 37°C for 60 minutes, and then terminated at 98°C for 2 minutes.

3. Detected with a microplate reader (excitation light 492 nm, emission light 515 nm), Figure 5A shows the probe gradient fluorescence value of the non-specific cleavage of the fluorescent probe by FnCas12a, the result shows that the fluorescence value is proportional to the amount of fluorescent probe.

4. The construction of the probe minimum detection line fluorescence value experiment of FnCas12a cleavage fluorescent probe, including the following steps:

1. In 8 groups of 100uL reaction systems, purified FnCas12a (250nM), guide RNA-pAcGFP1-N1-1 (250nM), pAcGFP1-N1 plasmid (4uL), fluorescent probe (FAM-TTATT-BHQ1) ( 10uM, 1uM, 0.1uM, 0.01uM, 0.001uM, 0.0001uM, 0.00001uM, 0.000001uM), RNase inhibitor 2uL, buffer NEBbuffer 3, and each group was repeated three times.

2. The reaction was carried out at 37°C for 60 minutes, and then terminated at 98°C for 2 minutes.

3. Detected with a microplate reader (excitation light 492nm, emission light 515nm), Figure 5B shows the lowest detection line fluorescence value of the probe for FnCas12a cleavage of the fluorescent probe, the results show that the probe concentration is too low will affect the fluorescence detection sensitivity, The lowest detection limit of the needle can reach 60pM.

5. The experimental construction for exploring the material influence of FnCas12a non-specifically cleaved fluorescent probes, including the following steps:

1. In eight groups of 100uL reaction systems, add purified FnCas12a (250nM), guide RNA-pAcGFP1-N1-1 (250nM), pAcGFP1-N1 plasmid (4uL), fluorescent probe (FAM- TTATT-BHQ1) (4uM), RNase inhibitor 2uL, buffer NEB buffer 3 to make up.

2. The reaction was carried out at 37°C for 60 minutes, and then terminated at 98°C for 2 minutes.

3. Detected with a microplate reader (excitation light 492nm, emission light 515nm), Figure 5C shows the fluorescence value of the material affected by the non-specific cleavage of the fluorescent probe by FnCas12a, the results show that FnCas12a protein The detection error of the fluorescence value in the blank experiment greater than that of guide RNA.

Embodiment 5: Construction and application of CRISPR cleavage reaction of hydrophobin-modified chip, including the following steps:

1. Same as step 1 in Example 1.

2. Same as step 2 of Example 1.

3. The experimental design of the chip substrate and hydrophobin modification, including the following steps:

1. The experimental process is shown in Figure 6. The chip substrate is modified with hydrophobin and dried in a ventilated place.

2. Add FnCas12a protein and guide RNA to air-dried hydrophobin for incubation, and then add specific substrate (pAcGFP1-N1 plasmid, pDsRed-Monomer-N1 plasmid) and fluorescent probe (FAM-TTATT-BHQ1), 37 °C reaction for 15min.

Fourth, the construction and application of the CRISPR cleavage reaction of the hydrophobin-modified chip, including the following steps:

1. Set up three groups of parallel experiments, and at the same time set up three groups of parallel experiments without hydrophobin modification. In each group of parallel experiments, a total of five 5ul reaction systems were set up, and purified FnCas12a was added to the pAcGFP1-N1 and pDsRed-Monomer-N1 experimental groups respectively. (250nM), guide RNAs—pAcGFP1-N1-1 (250nM), pDsRed-Monomer-N1-1 (250nM), pAcGFP1-N1 plasmid (0.75uL), pDsRed-Monomer-N1 plasmid (0.75uL), fluorescent probe Needle (FAM-TTATT-BHQ1) (1uM), RNase inhibitor 0.25uL, buffer NEB buffer 3 supplemented; pAcGFP1-N1, pDsRed-Monomer-N1 control groups exchanged the two crRNAs, and other experimental conditions remained unchanged ; In the negative control group, no FnCas12a protein was added, and other experimental conditions remained unchanged.

2. The results were observed under a fluorescence confocal microscope. Figure 7 shows the fluorescence detection results of the CRISPR cleavage reaction on the hydrophobin-modified and non-hydrophobin-modified chips. The results indicate that there is a high possibility of protein interaction between hydrophobin and FnCas12a protein. and enhance the cleavage ability of FnCas12a protein.

Development of a CRISPR high-throughput biochip method for detection of single-gene mutation diseases:

Using the cleavage properties of FnCas12a and the modification ability of hydrophobin, we have invented a CRISPR high-throughput biochip for the specific detection of single-gene mutation diseases. The chip has the characteristics of high efficiency, low cost, short cycle, strong specificity, and can detect The material is stable, the operation difficulty is low, and the results are intuitive and reliable.

The whole reaction system can be roughly divided into three steps, the first is the cleavage reaction of Cas12a protein, the second is the fluorescence reaction of the chip substrate modified by hydrophobin HFBⅠ, and the third is the high-throughput detection of HFBⅠ-immobilized Cas12a. For the cleavage reaction stage of Cas12a protein, three materials are the key to the experiment, namely Cas12a, crRNA and fluorescent probe. In addition to the FnCas12a protein used in the examples, other Cas12a proteins are also suitable for this method.

For the fluorescent reaction of the chip substrate modified by hydrophobin, the present invention selects HFBⅠ hydrophobin to modify the chip substrate and fix the Cas protein on the chip. After interacting with the FnCas12a protein, the ability of the Cas12a protein to drive and cut can be enhanced, and FAM is selected. As with BHQ1-labeled short ssDNA, other labeling methods that detect single-stranded cleavage are theoretically applicable, as long as the fluorescent probe is cleaved to produce a detectable difference.

We not only innovatively achieved the immobilization of CRISPR Cas protein by hydrophobin HFBⅠ, but also achieved high-throughput detection of single-gene diseases. On the premise of ensuring the biological activity of Cas protein, we use protein interaction and high-throughput detection to improve the efficiency of disease detection. The proposed methodology will have broad prospects and applications in gene mutation disease detection.

Table 1 Gene sequences involved in the present invention

Table 2 Names and GI numbers of FnCas12a protein and LbCas12a protein involved in the present invention

Although the preferred embodiments of the present invention have been described above with reference to the accompanying drawings, the present invention is not limited to the above-mentioned specific embodiments. Under the inspiration of the present invention, without departing from the spirit of the present invention and the protection scope of the claims, personnel can also make many forms, which all fall within the protection scope of the present invention.

<110> Tianjin University

<120> CRISPR high-throughput biochips for single-gene mutation detection

<130>

<160> 12

<170>

<210> 1

<211> 4726

<212> DNA

<213> Artificial sequences

<400> 1

tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60

cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120

gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180

atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240

aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300

catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360

catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420

atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480

ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540

acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta 600

ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg 660

gatccaccgg tcatggtgag caagggcgcc gagctgttca ccggcatcgt gcccatcctg 720

atcgagctga atggcgatgt gaatggccac aagttcagcg tgagcggcga gggcgagggc 780

gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcctgtg 840

ccctggccca ccctggtgac caccctgagc tacggcgtgc agtgcttctc acgctacccc 900

gatcacatga agcagcacga cttcttcaag agcgccatgc ctgagggcta catccaggag 960

cgcaccatct tcttcgagga tgacggcaac tacaagtcgc gcgccgaggt gaagttcgag 1020

ggcgataccc tggtgaatcg catcgagctg accggcaccg atttcaagga ggatggcaac 1080

atcctgggca ataagatgga gtacaactac aacgcccaca atgtgtacat catgaccgac 1140

aaggccaaga atggcatcaa ggtgaacttc aagatccgcc acaacatcga ggatggcagc 1200

gtgcagctgg ccgaccacta ccagcagaat acccccatcg gcgatggccc tgtgctgctg 1260

cccgataacc actacctgtc cacccagagc gccctgtcca aggaccccaa cgagaagcgc 1320

gatcacatga tctacttcgg cttcgtgacc gccgccgcca tcacccacgg catggatgag 1380

ctgtacaagt gagcggccgc gactctagat cataatcagc cataccacat ttgtagaggt 1440

tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc 1500

aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat 1560

cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact 1620

catcaatgta tcttaaggcg taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa 1680

atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata 1740

aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac 1800

tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc 1860

cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa 1920

atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg 1980

cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg 2040

tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag 2100

gtggcacttt tcggggaaat gtgcgcggaa cccctatttg ttattttttc taaatacatt 2160

caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 2220

ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa 2280

gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac 2340

caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa 2400

ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag 2460

ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc2520

cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt 2580

ttgcaaagat cgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat 2640

tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac 2700

agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc 2760

ttttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc 2820

tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag 2880

cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc 2940

ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg 3000

atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc 3060

ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc 3120

cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga 3180

cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca 3240

tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg 3300

atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg 3360

ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg 3420

gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga 3480

ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg 3540

gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta gggggaggct 3600

aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag 3660

acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg 3720

ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc 3780

cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg 3840

ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg atttaaaact 3900

tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 3960

cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 4020

ttcttgagat ccttttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 4080

accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 4140

cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca 4200

cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 4260

tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 4320

taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 4380

gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 4440

agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 4500

ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 4560

acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 4620

caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc 4680

tgcgttatcc cctgattctg tggataaccg tattaccgcc atgcat 4726

<210> 2

<211> 4691

<212> DNA

<213> Artificial sequences

<400> 2

tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60

cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120

gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180

atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240

aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300

catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360

catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420

atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480

ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540

acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta 600

ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg 660

gatccaccgg tcgccaccat ggacaacacc gaggacgtca tcaaggagtt catgcagttc 720

aaggtgcgca tggagggctc cgtgaacggc cactacttcg agatcgaggg cgagggcgag 780

ggcaagccct acgagggcac ccagaccgcc aagctgcagg tgaccaaggg cggccccctg 840

cccttcgcct gggacatcct gtccccccag ttccagtacg gctccaaggc ctacgtgaag 900

caccccgccg acatccccga ctacatgaag ctgtccttcc ccgagggctt cacctgggag 960

cgctccatga acttcgagga cggcggcgtg gtggaggtgc agcaggactc ctccctgcag 1020

gacggcacct tcatctacaa ggtgaagttc aagggcgtga acttccccgc cgacggcccc 1080

gtaatgcaga agaagactgc cggctgggag ccctccaccg agaagctgta cccccaggac 1140

ggcgtgctga agggcgagat ctcccacgcc ctgaagctga aggacggcgg ccactacacc 1200

tgcgacttca agaccgtgta caaggccaag aagcccgtgc agctgcccgg caaccactac 1260

gtggactcca agctggacat caccaaccac aacgaggact acaccgtggt ggagcagtac 1320

gagcacgccg aggcccgcca ctccggctcc cagtagagcg gccgcgactc tagatcataa 1380

tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc 1440

tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata 1500

atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc 1560

attctagttg tggtttgtcc aaactcatca atgtatctta aggcgtaaat tgtaagcgtt 1620

aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag 1680

gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt 1740

gttccagtttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga 1800

aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg 1860

gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct 1920

tgacgggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc 1980

gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt 2040

aatgcgccgc tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct 2100

atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga 2160

taaatgcttc aataatattg aaaaaggaag agtcctgagg cggaaagaac cagctgtgga 2220

atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa 2280

gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca 2340

gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc 2400

ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt 2460

ttttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag 2520

gaggcttttt tggaggccta ggcttttgca aagatcgatc aagagacagg atgaggatcg 2580

tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg 2640

ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg 2700

ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat 2760

gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca 2820

gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg 2880

gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat 2940

gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa 3000

catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg 3060

gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg 3120

cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg 3180

gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat 3240

caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac 3300

cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc 3360

cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc 3420

ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg 3480

gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt 3540

tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa 3600

cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg tcgtttgttc 3660

ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt 3720

ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag 3780

gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcctca ggttactcat 3840

atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc 3900

ttttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 3960

accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 4020

gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac 4080

caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc 4140

tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg 4200

ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt 4260

tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt 4320

gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc 4380

tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca 4440

gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata 4500

gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 4560

ggcggagcct atggaaaaac gccagcaacg cggcctttttt acggttcctg gccttttgct 4620

ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta 4680

ccgccatgca t 4691

<210> 3

<211> 43

<212> DNA

<213> Artificial sequences

<400> 3

cgtcaatggg tggagtattt acggatctac aacagtagaa att 43

<210> 4

<211> 43

<212> DNA

<213> Artificial sequences

<400> 4

agttccgcgt tacataactt acggatctac aacagtagaa att 43

<210> 5

<211> 42

<212> DNA

<213> Artificial sequences

<400> 5

gagtaacaga catggaccat cagatctaca acagtagaaa tt 42

<210> 6

<211> 324

<212> DNA

<213> Homo sapiens (Human)

<400> 6

ggtcgcgagt gcgttaattg cggggcaatg tctaccccac tgtggcgtcg tgacggtacc 60

ggtcattatc tgtgtaacgc ctgcggtctg taccataaaa tgaacggcat caaccgtccg 120

ctgattaaac cgcaacgtcg tctgtctgca agtcgtcgcg ttggtctgag ttgcgcaaat 180

tgtcaaacca ccaccaccac cctgtggcgt cgtaacgcag aaggcgaacc agtttgtaac 240

gcttgcggcc tgtatatgaa actgcacggc gttccacgtc cactggcaat gcgtaaagaa 300

ggcatccaga cccgcaaacg caaa 324

<210> 7

<211> 50

<212> DNA

<213> Homo sapiens (Human)

<400> 7

gtcacgccac ttgacaggcg agtaacagac atggaccatc aggaaacatt 50

<210> 8

<211> 55

<212> DNA

<213> Artificial sequences

<400> 8

gccggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagc 55

<210> 9

<211> 27

<212> DNA

<213> Artificial sequences

<400> 9

gtggcgataa gtcgtgtctt accgggt 27

<210> 10

<211> 28

<212> DNA

<213> Artificial sequences

<400> 10

gtcaaaccgc tatccacgcc cattgatg 28

<210> 11

<211> 25

<212> DNA

<213> Artificial sequences

<400> 11

gtacaaggcc aagaagcccg tgcag 25

<210> 12

<211> 32

<212> DNA

<213> Artificial sequences

<400> 12

gtctcttgat cgatctttgc aaaagcctag gc 32

Claims (9)

1. detecting the CRISPR high flux biochip of single gene mutation, which is characterized in that construction method includes the following steps:

(1) chemical synthesis contains the coli expression carrier of Cas12a protein sequence, specific substrate, containing PAM sequence PCR upstream primer, downstream primer, fluorescence probe, and corresponding to the crRNA of Cas12a and corresponding single gene mutation disease, really Determine expression of the FnCas12a albumen in Escherichia coli recombinant plasmid；

(2) compound of Cas12a and crRNA is to specific substrate --- and double-stranded DNA carries out the structure of the system of specific cutting Build, including to double-stranded DNA cutting experiment ratio optimization, to the cutting of pcr amplified fragment；

(3) compound of Cas12a and crRNA carries out the building of the system of Non-specific cleavage to single stranded DNA, including to common The cutting of single stranded DNA, the cutting to fluorescence probe single stranded DNA；

(4) chip base is subjected to hydrophobin modification；

(5) high-throughput chip base is constructed, including hydrophobin to the fixation of Cas12a-crRNA compound, compound to special Property substrate specificity cutting and to the Non-specific cleavage of fluorescence probe.

2. detecting the CRISPR high flux biochip of single gene mutation according to claim 1, which is characterized in that the step Suddenly (4) hydrophobin refers to some amphiphilic albumen, can pass through the both sexes for self being assembled to form nanometer grade thickness at two-phase interface Protein film.

3. detecting the CRISPR high flux biochip of single gene mutation according to claim 2, which is characterized in that the step Suddenly (4) hydrophobin refers to HGF I, HFB I.

4. detecting the CRISPR high flux biochip of single gene mutation according to claim 1, which is characterized in that described Cas12a albumen is preferably FnCas12a albumen.

5. detecting the construction method of the CRISPR high flux biochip of single gene mutation, which comprises the steps of: (1) chemical synthesis contains on the coli expression carrier, specific substrate, the PCR containing PAM sequence of Cas12a protein sequence Primer, downstream primer, fluorescence probe are swum, and corresponding to the crRNA of Cas12a and corresponding single gene mutation disease, is determined Expression of the FnCas12a albumen in Escherichia coli recombinant plasmid；

(2) compound of Cas12a and crRNA is to specific substrate --- and double-stranded DNA carries out the structure of the system of specific cutting Build, including to double-stranded DNA cutting experiment ratio optimization, to the cutting of pcr amplified fragment；

(3) compound of Cas12a and crRNA carries out the building of the system of Non-specific cleavage to single stranded DNA, including to common The cutting of single stranded DNA, the cutting to fluorescence probe single stranded DNA；

(4) chip base is subjected to hydrophobin modification；

(5) high-throughput chip base is constructed, including hydrophobin to the fixation of Cas12a-crRNA compound, compound to special Property substrate specificity cutting and to the Non-specific cleavage of fluorescence probe.

6. detecting the construction method of the CRISPR high flux biochip of single gene mutation, feature according to claim 5 It is, step (4) hydrophobin refers to some amphiphilic albumen, can be at two-phase interface by self being assembled to form nanoscale The Amphiphilic proteins film of thickness.

7. detecting the construction method of the CRISPR high flux biochip of single gene mutation, feature according to claim 6 It is, step (4) hydrophobin refers to HGF I, HFB I.

8. detecting the construction method of the CRISPR high flux biochip of single gene mutation, feature according to claim 5 It is, the Cas12a albumen is preferably FnCas12a albumen.

9. detecting the application of the CRISPR high flux biochip of single gene mutation described in claim 1.