CN114231596B - Gene detection method based on CRISPR/dcas9 and magnetic nano material and application thereof - Google Patents

Gene detection method based on CRISPR/dcas9 and magnetic nano material and application thereof Download PDF

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CN114231596B
CN114231596B CN202111513257.3A CN202111513257A CN114231596B CN 114231596 B CN114231596 B CN 114231596B CN 202111513257 A CN202111513257 A CN 202111513257A CN 114231596 B CN114231596 B CN 114231596B
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王玉鑫
陈明慧
汤武玥
鲁晓桐
周昊
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Xuzhou Medical University
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Abstract

A gene detection method based on CRISPR/dcas9 and magnetic nano materials comprises the following steps: acquiring a target oncogene sequence to be detected, and selecting specific sgRNA; preparing streptavidin magnetic nano-microspheres, then reacting the streptavidin magnetic nano-microspheres with SNAP-Biotin, and then combining with SNAP-tag on CRISPR/dCAS9 protein to form CRISPR@magnetic nano-microspheres; incubating the prepared CRISPR@magnetic nanospheres with the sgRNA selected in the step (1), adding the incubated CRISPR@magnetic nanospheres into a body fluid sample to be detected, and adding a nucleic acid dye, wherein when a target oncogene exists in the body fluid sample to be detected, the nucleic acid dye generates a fluorescence detection signal on the surface of the magnetic nanospheres, so that the detection of the target oncogene is realized. The invention adopts the method of combining CRISPR/dcas9 technology and magnetic nano-microspheres, so that the body fluid sample can be rapidly separated, the sensitivity is increased, the signal is amplified, and the detection specificity is improved. The specificity of the detection of the breast cancer related genes is strong, the molecular stability is good, the detection cost is low, and the problem of poor specificity in the existing detection method is solved.

Description

Gene detection method based on CRISPR/dcas9 and magnetic nano material and application thereof
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to detection and application of CRISPR/dcas9 combined magnetic nano-microspheres to breast cancer related genes.
HER-2/neu is an oncogene and is closely related to the occurrence and development of breast cancer. HER-2/neu gene-related protein amplification and overexpression occurs at about 0%, 22.5%, 46.7% in female breast simple hyperplasia, atypical hyperplasia, and breast cancer, with primary invasive breast cancer of 22% -29%, acne ductal carcinoma in situ of almost 100%, and a large percentage of ductal carcinoma in situ of the breast. Therefore, the HER-2/neu gene protein has important clinical value for early diagnosis of female breast cancer. By detecting the gene, early screening of breast cancer can be realized.
The CRISPR/Cas9 complex is a novel DNA-targeted editing tool consisting of clustered, regularly interspaced short palindromic repeats and CRISPR-associated protein mutants, an adaptive immune system that is widely present in most bacteria and archaebacteria. Guide RNAs (sgrnas) in CRISPR/Cas systems can recognize and bind exogenous nucleic acid molecules, enabling subsequent splicing editing of related genes. The dCas9 protein is a mutant of the Cas9 protein, that is, two nuclease active regions RuvC1 and HNH of Cas9 endonuclease are simultaneously mutated, so that the endonuclease activity of the Cas9 protein is completely disappeared, and only the ability of being guided and recognized into a genome by sgrnas is reserved, and the target nucleic acid is specifically bound and captured. By utilizing the specific sequence recognition characteristic of the CRISPR/dCAS system, the detection sensitivity and the specificity are obviously improved when the CRISPR/dCAS system is applied to nucleic acid detection.
Biomagnetic separation is one of the most cost-effective methods in the field of bioseparation technology, and has played an increasingly important role in many bioscience studies. By modifying different chemical groups on the surface of the magnetic beads, the magnetic beads are effectively connected with ligands with biological affinity. Can be used for rapidly and simply separating biological molecules when an external magnetic field is applied. For example, avidin or antibodies are modified on magnetic beads, which bind to their high affinity target substances, which may be nucleic acids, proteins, bacteria, viruses, cells, etc. In addition, the biological magnetic separation technology is adopted, the defects that the centrifugation step is time-consuming and labor-consuming and the sample treatment link is complex and troublesome in the traditional separation method are eliminated, and the whole experimental process operation becomes simpler and quicker.
Disclosure of Invention
The invention aims to provide a method for realizing the portable rapid hybridization and detection of breast cancer related genes based on a CRISPR/dcas9 magnetic nanotechnology, which combines the CRISPR/dcas9 technology with the magnetic nanotechnology to rapidly and specifically detect breast cancer HER-2/neu genes so as to realize early screening and prognosis monitoring of breast cancer.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
a gene detection method based on CRISPR/dcas9 and magnetic nano materials comprises the following steps:
(1) And acquiring a target oncogene sequence to be detected, selecting specific sgrnas according to the difference between the pairing Tm temperature value and the delta G energy value, evaluating the specificity of the sgrnas, and selecting the sgrnas with the best specificity.
(2) Preparing streptavidin magnetic nano-microsphere, then reacting the streptavidin magnetic nano-microsphere with SNAP-Biotin, and then combining with SNAP-tag on CRISPR/dCAS9 protein to form CRISPR@magnetic nano-microsphere.
(3) Incubating the prepared CRISPR@magnetic nanospheres with the sgRNA selected in the step (1), adding the incubated CRISPR@magnetic nanospheres into a body fluid sample to be detected, and adding a nucleic acid dye, wherein when a target oncogene exists in the body fluid sample to be detected, the nucleic acid dye generates a fluorescence detection signal on the surface of the magnetic nanospheres, so that the detection of the target oncogene is realized.
Further, the preparation method of the streptavidin magnetic nanoparticle comprises the following steps:
superparamagnetism oil-soluble magnetic nano particles are prepared by adopting a high-temperature oil phase reduction method, and the prepared magnetic nano particles are used as magnetic cores to form reverse microemulsion by a water-in-oil method to prepare SiO (silicon dioxide) 2 The magnetic nanometer microsphere is provided with a shell and has a core-shell structure; the prepared magnetic nano microsphere is pretreated by using an aminoethylaminopropyl polydimethylsiloxane buffer solution, and then is wrapped by using streptavidin to form the streptavidin magnetic nano microsphere.
Further, the oncogene of interest is HER-2/neu.
Further, the step (1) includes:
the sequence of HER-2/neu gene is obtained, specific sgRNA is selected according to the difference of the pairing Tm temperature value and the delta G energy value, wherein the pairing Tm temperature value of the target gene is 54.4 ℃, the delta G energy value is 0.33kcal/mole, the sgRNA with the Tm value higher than 54.4 ℃ and the delta G energy value lower than 0.33kcal/mole in the pairing region of the target gene is selected, the specificity of the selected sgRNA is evaluated, and the sgRNA with the best specificity is selected.
The invention also provides application of the method in preparation of a breast cancer detection kit or breast cancer detection equipment.
The invention also provides a detection kit which comprises the CRISPR@magnetic nanoparticle prepared by the invention and selected sgRNA.
Compared with the prior art, the invention has the beneficial technical effects that:
1. the method has the advantages of strong specificity for detecting the breast cancer related genes, good molecular stability and low detection cost, and solves the problem of poor specificity in the existing detection method.
2. The method of combining CRISPR/dcas9 technology and magnetic nano-microsphere is adopted, so that the body fluid sample is rapidly separated, the sensitivity is increased, the signal is amplified, and the detection specificity is improved.
Drawings
FIG. 1 is a graph of the results of CRISPR/dCAS9 electrophoresis of sgRNA against the HER-2 gene;
FIG. 2 is a magnetic nanoparticle electron microscope characterization diagram;
FIG. 3 is a schematic diagram of a nucleic acid capture detection;
FIG. 4 is a graph showing the results of CRISPR@magnetic nanoparticle binding to HER-2 gene of different contents.
Detailed Description
The invention will be further illustrated in the following examples, but is not limited thereto.
The 1 x PBS configuration was: weighing 137mmol/LNaCl,2.7mmol/L KCl,10mmpl/L Na 2 HPO 4 ，1.8mmol/L KH 2 PO 4 Dissolved in 800mL of water. The pH was adjusted to 7.4 and then fixed to 1L.
10×aeaps buffer:20mM Tris-HCl (pH 7.5), 150mM NaCl, 1mM disodium ethylenediamine tetraacetate (Na) 2 EDTA), 1mM ethylenediamine tetraacetic acid (EGTA), 1% Triton, 2.5mM sodium pyrophosphate, 1mM beta-glycerophosphate, 1mM Na 3 VO 4 1 μg/mL leupeptin. CST suggests the addition of 1mM protease inhibitor (PMSF) immediately prior to use.
The primer and DNA and RNA sequences used in the kit are synthesized by Shanghai.
A method for realizing portable rapid hybridization and detection of breast cancer related genes based on CRISPR/dcas9 magnetic nanotechnology comprises the following steps:
(1) Design of high-specificity sgrnas: according to the characteristics of the breast cancer amplification target sequence, the possible sgRNA sites (PAM sequence is TGG) are analyzed by using an online website (http:// crispr. Mit. Edu /), and the optimal sgRNA base sequence is screened. Design-specific sgrnas were selected based on differences in paired Tm temperature values and Δg energy values. The Tm value of the target gene pairing is 54.4 ℃ and the DeltaG energy value is 0.33kcal/mole, so that the Tm value of the pairing area of the designed sgRNA and the target gene is higher than 54.4 ℃ and the DeltaG energy value is lower than 0.33kcal/mole, and the prepared sgRNA can be efficiently and specifically combined with the target HER-2/neu gene. The specificity of the sgrnas was evaluated and the sgrnas with the best specificity were selected (fig. 1). Designed within the range of about 80-150bp upstream and downstream of the selected sgRNA core sequence, the designed dCAS9 protein is subjected to SNAP-tag labeling during the expression process of the dCAS9 protein.
HER-2/neu gene (SEQ ID No. 1): AH002823.2, bolded as PAM site, underlined as sgRNA targeting region:
dCAS9-sgRNA Gene sequence (SEQ ID No. 2): the sequence at the streak is the target gene pairing region GGAGACTTC GTTGGAATGCAGTAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU
(2) Preparing affinity magnetic nano-microspheres: weigh 2.8X10 -3 -2.9×10 -3 The ferric acetylacetonate with mol/L and 15-25mL benzyl alcohol are fully mixed in an anaerobic environment, transferred into a Teflon cup, finally transferred into a high-pressure reaction kettle and sealed for reaction for 2-6h at 170-180 ℃. And taking out the reaction liquid after the reaction is finished, and centrifuging in a centrifuge to remove the supernatant to obtain the oil-soluble magnetic nano particles. TitonX-100 and n-hexanolAnd the cyclohexane is uniformly mixed according to different proportions to form a transparent and stable microemulsion system. Then adding a proper amount of Fe 3 O 4 After ultrasonic treatment for 10-40min, 1-3mL of concentrated ammonia water and 1-3mL of tetraethoxysilane are added, and stirring is carried out for 1-3h at 30-50 ℃. Standing for precipitation, cleaning with ethanol, and calcining at 400-800 ℃ for 2-4h to obtain the amino-modified magnetic nanoparticle. 1mg of the magnetic nano particles prepared by the method are added into a certain amount of mixed solution of methanol and glycerol for ultrasonic treatment for 10-40min. Adding a certain amount of AEAPS into the mixed solution, uniformly mixing by ultrasonic, reacting for 1-3h at 80-100 ℃, taking out particles, drying the particles in vacuum for 1-2h by using methanol, and collecting the particles. Then wrapping with streptavidin, preparing PBS buffer solution with RNase enzyme-removed water, and autoclaving to obtain sterile PBS. 0.5-1.5mg of streptavidin was dissolved in 0.5mL of PBS and dispensed into EP tubes at 50. Mu.L per tube. 5-7mg of the treated and modified amino particles were added to 1mL of PBS and dispersed by sonication. 500. Mu.L of the modified amino group particle PBS suspension was added to 50. Mu.L of the above streptavidin solution, and the reaction was carried out at room temperature with shaking. After the reaction was complete, the particles were dispersed in 1mL of PBS solution and stored at 4 ℃ for use. FIG. 2 is a diagram showing the characterization of the prepared magnetic nanoparticle.
(3) Preparing a CRISPR@magnetic nanoparticle probe and detecting a breast cancer related gene:
as shown in fig. 3, the prepared affinity magnetic nanoparticle is utilized to construct a crispr@magnetic nanotechnology to realize detection of a target gene. 1-5 mu L of dCAS9 protein with the concentration of 1-5 mu mol/L is taken, 1-5 mu L of SNAP-Biotin with the concentration of 1-5 mu mol/L and 10-50 mu L of avidinated magnetic nano microsphere with the concentration of 1-2mg/mL are added, and the reaction is carried out for 20-50min at the temperature of 25-37 ℃. Because the CRISPR/dCAS9 protein is provided with the SNAP label, the CRISPR/dCAS 9-SNAP-Biotin-magnetic nanoparticle (CRISPR@magnetic nanoparticle detection probe) is prepared. And adding 0.5-5 mu L of guide RNA (sgRNA) with the concentration of 0.5-4 mu mol/L, complementarily pairing the sgRNA with a HER-2/neu gene breast cancer mutant fragment, reacting for 20-50min at the temperature of 25-37 ℃, so as to quickly, sensitively and specifically bind a target gene to the surface of a CRISPR@magnetic nanoparticle detection probe, collecting fluorescent signals on the surface of the magnetic nanoparticle through the steps of magnetic separation, washing and the like, wherein the fluorescent intensity is positively correlated with the content of the target gene, and the CRISPR@magnetic nanoparticle shows fluorescent display of different degrees when being bound with HER-2 with different contents. Thereby realizing the early screening detection and prognosis monitoring of the HER-2/neu gene of the breast cancer.
Example 1
Weigh 2.83×10 -3 The mol/L ferric acetylacetonate and 20mL benzyl alcohol are fully mixed in an anaerobic environment, transferred into a Teflon cup, finally transferred into a high-pressure reaction kettle, and sealed and reacted for 2 hours at 170 ℃. And taking out the reaction liquid after the reaction is finished, and centrifuging in a centrifuge to remove the supernatant to obtain the oil-soluble magnetic nano particles. The TitonX-100, the n-hexanol and the cyclohexane are uniformly mixed according to the proportion of 1:2:4 to form a transparent and stable microemulsion system. Then adding a proper amount of Fe 3 O 4 After ultrasonic treatment for 20min, 2mL of concentrated ammonia water and 2mL of ethyl orthosilicate were added and stirred at 30℃for 2h. Standing for precipitation, cleaning with ethanol, and calcining at 500 ℃ for 2 hours to obtain the amino-modified magnetic nano particles. 1mg of the magnetic nanoparticle prepared by the method is added into a certain amount of mixed solution of methanol and glycerol for ultrasonic treatment for 30min. Adding a certain amount of AEAPS into the mixed solution, uniformly mixing by ultrasonic, reacting for 2 hours at 90 ℃, taking out particles, drying the particles in vacuum with methanol for 1 hour, and collecting the particles. Then wrapping with streptavidin, preparing PBS buffer solution with RNase enzyme-removed water, and autoclaving to obtain sterile PBS. 1mg of streptavidin was dissolved in 0.5mL of PBS, and the mixture was dispensed into EP tubes at a concentration of 50. Mu.L per tube. 5mg of the treated and amino-modified particles were added to 1mL of PBS and dispersed by sonication. 500. Mu.L of the modified amino group particle PBS suspension was added to 50. Mu.L of the above streptavidin solution, and the reaction was carried out at room temperature with shaking. After the reaction was complete, the particles were dispersed in 1mL of PBS solution and stored at 4 ℃ for use.
Example 2
Weigh 2.81×10 -3 The mol/L ferric acetylacetonate and 20mL benzyl alcohol are fully mixed in an anaerobic environment, transferred into a Teflon cup, finally transferred into a high-pressure reaction kettle, and sealed and reacted for 2 hours at 170 ℃. Taking out the reaction solution after the reaction is finished, centrifuging in a centrifuge to remove the supernatant to obtain oil-soluble productMagnetic nanoparticles. The TitonX-100, the n-hexanol and the cyclohexane are uniformly mixed according to the proportion of 1:2:5 to form a transparent and stable microemulsion system. Then adding a proper amount of Fe 3 O 4 After ultrasonic treatment for 20min, 1mL of concentrated ammonia water and 2mL of ethyl orthosilicate were added and stirred at 30℃for 2h. Standing for precipitation, cleaning with ethanol, and calcining at 500 ℃ for 2 hours to obtain the amino-modified magnetic nano particles. 1mg of the magnetic nanoparticle prepared by the method is added into a certain amount of mixed solution of methanol and glycerol for ultrasonic treatment for 30min. Adding a certain amount of AEAPS into the mixed solution, uniformly mixing by ultrasonic, reacting for 2 hours at 100 ℃, taking out particles, drying the particles in vacuum for 1 hour by using methanol, and collecting the particles. Then wrapping with streptavidin, preparing PBS buffer solution with RNase enzyme-removed water, and autoclaving to obtain sterile PBS. 1.5mg of streptavidin was dissolved in 0.5mL of PBS, and the mixture was dispensed into EP tubes at a concentration of 50. Mu.L per tube. 5mg of the treated and amino-modified particles were added to 1mL of PBS and dispersed by sonication. 500. Mu.L of the modified amino group particle PBS suspension was added to 50. Mu.L of the above streptavidin solution, and the reaction was carried out at room temperature with shaking. After the reaction was complete, the particles were dispersed in 1mL of PBS solution and stored at 4 ℃ for use.
Example 3
Six experiments were designed, two blank groups, four experimental groups. 1 mu L of dCAS9 protein with the concentration of 1 mu mol/L is taken, 1 mu L of SNAP-Biotin with the concentration of 1 mu mol/L and 10 mu L of avidinated magnetic nano-microsphere with the concentration of 1mg/mL are added, and the mixture is reacted for 25min at 28 ℃, so that CRISPR/dCAS 9-SNAP-Biotin-magnetic nano-microsphere (CRISPR@magnetic nano-microsphere) is formed, and after grouping, 1 mu L of three groups of guide RNAs (sgRNAs) with different concentrations of 1 mu mol/L are added. And 6 PCR reaction tubes are taken, the reaction liquids are respectively packaged into the PCR tubes, no genes to be detected are not added into the No.1 and No.2 centrifuge tubes, target gene fragments corresponding to different concentrations are sequentially and incrementally added into the No. 3, no. 4, no. 5 and No. 6 centrifuge tubes, the reaction is carried out for 25min at 28 ℃, the target gene fragments complementary to sgRNA are quickly, sensitively and specifically combined to the surfaces of CRISPR@magnetic nano microspheres, and fluorescent signals on the surfaces of the magnetic nano spheres are collected through the steps of magnetic separation, washing and the like, wherein the fluorescent intensity is positively correlated with the content of the target genes. Through the design of sgRNA, the early screening detection and prognosis monitoring of the HER-2/neu gene of the breast cancer are realized.
Example 4
Six experiments were designed, two blank groups, four experimental groups. 1 mu L of dCAS9 protein with the concentration of 2 mu mol/L is taken, 1 mu L of SNAP-Biotin with the concentration of 1 mu mol/L and 20 mu L of avidinated magnetic nano-microsphere with the concentration of 1mg/mL are added for reaction at 37 ℃ for 30min, so that CRISPR/dCAS 9-SNAP-Biotin-magnetic nano-microsphere (CRISPR@magnetic nano-microsphere) is formed, and after grouping, 1 mu L of three groups of guide RNAs (sgRNAs) with different concentrations of 2 mu mol/L are added. And 6 PCR reaction tubes are taken, the reaction liquids are respectively packaged into the PCR tubes, no genes to be detected are not added into the No.1 and No.2 centrifuge tubes, target gene fragments corresponding to different concentrations are sequentially and incrementally added into the No. 3, no. 4, no. 5 and No. 6 centrifuge tubes, and the reaction is carried out for 30min at 37 ℃, so that the target genes corresponding to sgRNA are rapidly, sensitively and specifically combined to the surfaces of CRISPR@magnetic nano-microspheres, and fluorescent signals on the surfaces of the magnetic nano-spheres are collected through the steps of magnetic separation, washing and the like, wherein the fluorescent intensity is positively correlated with the content of the target genes. Through the design of sgRNA, the early screening detection and prognosis monitoring of the HER-2/neu gene of the breast cancer are realized.
Example 5
Six experiments were designed, two blank groups, four experimental groups. 1 mu L of dCAS9 protein with the concentration of 3 mu mol/L is taken, 5 mu L of SNAP-Biotin with the concentration of 3 mu mol/L and 20 mu L of avidinated magnetic nano-microsphere with the concentration of 1mg/mL are added, and the mixture is reacted for 40min at 37 ℃ to prepare CRISPR/dCAS 9-SNAP-Biotin-magnetic nano-microsphere (CRISPR@magnetic nano-microsphere), and after grouping, 1 mu L of three groups of guide RNAs (sgRNAs) with different concentrations of 1 mu mol/L are added. And 6 PCR reaction tubes are taken, the reaction liquids are respectively packaged into the PCR tubes, no genes to be detected are not added into the No.1 and No.2 centrifuge tubes, target gene fragments corresponding to different concentrations are sequentially and incrementally added into the No. 3, no. 4, no. 5 and No. 6 centrifuge tubes, and the reaction is carried out for 25min at the temperature of 28 ℃, so that the target genes corresponding to sgRNA are rapidly, sensitively and specifically combined to the surfaces of CRISPR@magnetic nano microspheres, and fluorescent signals on the surfaces of the magnetic nano spheres are collected through the steps of magnetic separation, washing and the like, wherein the fluorescent intensity is positively correlated with the content of the target genes. Through the design of sgRNA, the early screening detection and prognosis monitoring of the HER-2/neu gene of the breast cancer are realized.
Example 6
Six experiments were designed, two blank groups, four experimental groups. 3 mu L of dCAS9 protein with the concentration of 1 mu mol/L is taken, 1 mu L of SNAP-Biotin with the concentration of 1 mu mol/L and 10 mu L of avidinated magnetic nano-microsphere with the concentration of 2mg/mL are added for reaction at 37 ℃ for 30min, so that CRISPR/dCAS 9-SNAP-Biotin-magnetic nano-microsphere (CRISPR@magnetic nano-microsphere) is formed, and 3 mu L of three groups of guide RNAs (sgRNAs) with different concentrations of 1 mu mol/L are added after grouping. And 6 PCR reaction tubes are taken, the reaction liquids are respectively packaged into the PCR tubes, no genes to be detected are not added into the No.1 and No.2 centrifuge tubes, target gene fragments corresponding to different concentrations are sequentially and incrementally added into the No. 3, no. 4, no. 5 and No. 6 centrifuge tubes, and the reaction is carried out for 30min at 37 ℃, so that the target genes corresponding to sgRNA are rapidly, sensitively and specifically combined to the surfaces of CRISPR@magnetic nano-microspheres, and fluorescent signals on the surfaces of the magnetic nano-spheres are collected through the steps of magnetic separation, washing and the like, wherein the fluorescent intensity is positively correlated with the content of the target genes. Through the design of sgRNA, the early screening detection and prognosis monitoring of the HER-2/neu gene of the breast cancer are realized.
Sequence listing
<110> Xuzhou university of medical science
<120> CRISPR/dcas9 and magnetic nanomaterial-based gene detection method and application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 757
<212> DNA
<213> HER-2/neu Gene sequence (HER-2/neu)
<400> 1
cccgggggtc ctggaagcca caaggtaaac acaacacatc cccctccttg actatgcaat 60
tttactagag gatgtggtgg gaaaaccatt atttgatatt aaaacaaata ggcttgggat 120
ggagtaggat gcaagctccc caggaaagtt taagataaaa cctgagactt aaaagggtgt 180
taagagtggc agcctaggga atttatcccg gactccgggg gagggggcag agtcaccagc 240
ctctgcattt agggattctc cgaggaaaag tgtgagaacg gctgcaggca acccaggcgt 300
cccggcgcta ggagggacga cccaggcctg cgcgaagaga gggagaaagt gaagctggga 360
gttgccgact cccagacttc gttggaatgc agttggaggg ggcgagctgg gagcgcgctt 420
gctcccaatc acaggagaag gaggaggtgg aggaggaggg ctgcttgagg aagtataaga 480
atgaagttgt gaagctgaga ttcccctcca ttgggaccgg agaaaccagg ggagcccccc 540
gggcagccgc gcgccccttc ccacggggcc ctttactgcg ccgcgcgccc ggcccccacc 600
cctcgcagca ccccgcgccc cgcgccctcc cagccgggtc cagccggagc catggggccg 660
gagccgcagt gagcaccatg gagctggcgg ccttgtgccg ctgggggctc ctcctcgccc 720
tcttgccccc cggagccgcg agcacccaag gtgggtc 757
<210> 2
<211> 96
<212> DNA/RNA
<213> HER-2/neu Gene sequence (dCAS 9-sgRNA)
<400> 2
ggagacttcg ttggaatgca gtagagcuag aaauagcaag uuaaaauaag gcuaguccgu 60
uaucaacuug aaaaaguggc accgagucgg ugcuuu 96

Claims (3)

1. A reagent for detecting breast cancer is characterized by comprising CRISPR@magnetic nano microspheres and sgRNA, wherein the sequence of the sgRNA is shown as SEQ ID No. 2.
2. A kit for detecting breast cancer, comprising the reagent of claim 1.
3. An apparatus for detecting breast cancer, comprising the reagent of claim 1.
CN202111513257.3A 2021-12-12 2021-12-12 Gene detection method based on CRISPR/dcas9 and magnetic nano material and application thereof Active CN114231596B (en)

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