CN115948405A - Visual circular RNA rapid detection kit based on CRISPR-Cas13a system and application thereof - Google Patents
Visual circular RNA rapid detection kit based on CRISPR-Cas13a system and application thereof Download PDFInfo
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Abstract
The invention provides a guide RNA optimized by sequence screening, which has strong pertinence and high anti-interference capability, can efficiently identify lung cancer fusion circular RNA and activate Cas13 to cut off a fluorescent reporter molecule, and realizes sensitive fluorescent detection; the kit for detecting F-circEA designed based on the guide RNA can effectively carry out lung cancer liquid biopsy detection, and has the advantages of high speed, low cost, low environmental use requirement and wide application prospect.
Description
Technical Field
The invention belongs to the field of detection reagents, and particularly relates to a visualized annular RNA rapid detection kit based on a CRISPR-Cas13a system and application thereof.
Background
Lung cancer is one of the most harmful malignant tumors to human health and life at present, patients are often diagnosed in an advanced stage or have metastasized, and the 5-year survival rate is only very low. The fusion gene plays an important role in the occurrence and development process of lung cancer, wherein the N-terminal part of the protein coded by the echinoderm microtubule-associated protein-like 4 (EML 4) is fused to the intracellular tyrosine kinase domain of Anaplastic Lymphoma Kinase (ALK) and is rearranged into EML4-ALK, so that the abnormal tyrosine kinase expression is caused, the cells are promoted to be continuously proliferated, and the cells are cancerized. At present, research reports that a novel F-circulating EA generated by lung cancer EML4-ALK fusion gene has a proliferation promoting effect, and tumor tissues and blood plasma of NSCLC patients contain F-circulating EA, which is an important clinical value of a biomarker for diagnosing EML4-ALK positive NSCLC patients (Shuanggyan Tan, Q.G., wenchen Pu, chenglin Guo, yun Yang, ke Wu, yaxin Liu, lunxu Liu, yu-query i and Yong Pen, circular RNA F-circulating free from EML4-ALK fusion gene as a non-level living biology for non-cell lung cancer cell, cell 2018), but no good method is used for detecting and limiting the clinical application of the F-circulating gene.
At present, the clinical preliminary diagnosis of the lung cancer fusion gene is mainly carried out by sequence analysis and immunohistochemistry through fluorescence real-time quantitative PCR (RT-PCR) or second-generation sequencing, but the methods have the defects of long detection time and period, high environmental requirement, high technical force requirement, high hardware equipment requirement, great psychological and physical trauma brought to patients by invasive sampling and the like, and the circRNA is an ideal candidate of a liquid biopsy biomarker, which greatly promotes the early diagnosis and early detection of the lung cancer. Therefore, there is an urgent need to develop a fast, low-cost method for detecting nucleic acid in liquid biopsy.
In recent years, the great potential of CRISPR systems in nucleic acid detection has been uncovered thanks to the feature that Cas12, cas13 and Cas14 exhibit strong trans-cleavage activity after recognizing and cleaving specific targets. Namely, the CRISPR-associated (Cas) protein can be used for recognizing a target sequence, simultaneously activating the trans-cutting of the Cas protein, and cleaving a DNA or RNA fluorescent reporter gene to realize fluorescent visual detection. For example, chinese patent No. CN111850097B discloses a nucleic acid detection system with a third-level signal amplification system (reporter magnetic bead) introduced on the basis of two rounds of signal amplification for nucleic acid amplification and nonspecific arbitrary cleavage of Cas protein, and has high detection sensitivity.
However, for F-circEA as a specific circular RNA, development of a method with higher sensitivity and detection accuracy based on the trans-cleavage activity of Cas protein is still to be further explored.
Disclosure of Invention
The invention aims to provide a visualized annular RNA rapid detection kit based on a CRISPR-Cas13a system and application thereof.
The invention provides a guide RNA, the sequence of which is shown in SEQ ID NO. 1.
The invention also provides application of the guide RNA in preparation of a lung cancer detection kit.
The invention also provides a circular RNA detection kit which comprises the guide RNA.
Further, the kit further comprises a Cas13a protein and a fluorescent reporter molecule;
the fluorescent reporter molecule is ssRNA with two ends respectively modified with a fluorescent group and a quenching group, and the sequence is shown as SEQ ID NO. 2.
Further, the above-mentioned fluorescent group is: FAM, cy3, cy5, HEX, or ROX; the quencher group is: BHQ1, BHQ2, BHQ3 or TAMRA.
Further, the fluorescent group is FAM and the quencher group is BHQ1.
The invention also provides application of the kit in preparing a lung cancer diagnostic reagent.
Further, the lung cancer diagnostic reagent is a reagent for detecting a lung cancer biomarker in blood.
Further, the above biomarker for lung cancer is a lung cancer fused circular RNA: F-circEA.
The invention has the beneficial effects that: according to the invention, through the design of the guide RNA and the fluorescent reporter molecule, the guide RNA with strong pertinence and high anti-interference capability is synthesized, the lung cancer fused cyclic RNA can be efficiently identified, the Cas13 is activated to cut off the fluorescent reporter molecule, and sensitive fluorescent detection is realized; the kit for detecting F-circEA based on the guide RNA design can effectively carry out lung cancer liquid biopsy detection, and has the advantages of rapidness, low cost, low environmental use requirement and wide application prospect.
The basic detection principle of the invention is as follows: when the guide RNA is used for targeted recognition of the lung cancer fused circular RNA, cas13a is activated, the fluorescence quenching fluorescent reporter molecule is cut off, and a fluorescent signal is generated.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 is a graph showing the results of the detection sensitivity of the fused circular RNA system in cells according to the present invention.
FIG. 2 is a diagram showing the results of the detection specificity of the fused circular RNA system in the cells of the present invention.
FIG. 3 is a diagram showing the optimized result of detecting fused circular RNA crRNA in the present invention.
Detailed Description
The raw materials and equipment used in the invention are known products, and are obtained by purchasing products sold in the market.
Example 1 use of guide RNAs and kits of the present invention for detection of circular RNAs
1. Test sample
(1) In vitro transcribing the artificially synthesized RNA sequence of the circular RNA cyclization site;
(2) Spiked in vitro transcribed RNA in total RNA of the blood sample;
(3) Fused circular RNA transfected in 293T, H1299 cells;
(4) Circular RNA in H2228 cells.
Wherein the sample (1) is double-stranded DNA synthesized by a gene synthesis method, and the sequence of the double-stranded DNA is as follows: (SEQ ID NO. 3):
GAAATTAATACGACTCACTATAGGGTGCAAGTGGCTGTGAAGACGCTGCCTGAAGTGTGCTCTGAAAATTCGAGCATCACCTTCTCCCCAGCCCTCTTCACAACC
sample (2) is prepared by adding the product of (1) to total RNA in blood;
sample (3) was PCR amplified and the linear F-circEA sequence was cloned into pcDNA3.0, complementary flanking sequences were added at both ends of the sequence, and transfection of 293T and H1299 was detected by inverse PCR, resulting in correct expression of the circular F-circEA.
The sample (4) is obtained by directly taking H2228 cultured cells.
2. Sequence to be detected (SEQ ID NO. 4)
TGCAAGTGGCTGTGAAGACGCTGCCTGAAGTGTGCTCTGAAAATT CGAGCATCACCTTCTCCCCAGCCCTCTTCACAACC
3. Isothermal amplification primer synthesis
RPA-F primer (SEQ ID NO. 5):
GAAATTAATACGACTCACTATAGGGTGCAAGTGGCTGTGAAGACG CTG
RPA-R primer (SEQ ID NO. 6): GGTTGTGAAGAGGGCTGGAGAA
4. Design and Synthesis of gRNAs
F-circEA-gRNA(SEQ ID NO.1)：
5’-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAAC GUGAUGCUCGAAUUUUCAGAGCACCACU-3’
5. Fluorescent reporter design and Synthesis
The ssRNA-FQ is an oligonucleotide molecule with 5 'end marked with FAM fluorescent group and 3' end marked with BHQ1 quenching group, and the specific sequence is (SEQ ID NO. 2) 5'-FAM-AAUGGCAAAUGGCA-BHQ1-3'.
6. Cell sample processing
The cells transfected by F-circEA from 6cm dishes were collected, and total RNA of H2228 and H1299 cells transfected by F-circEA was extracted using a commercial nucleic acid release kit (P073, novoxaz) and a self-made nucleic acid release agent, and treated at room temperature for 10min.
7. Isothermal amplification (RT-RPA)
RPA kit: twist Amp Basic
Brand name: twist Dx
Using the RNA standard near the transcribed and purified F-circRNA fusion site as a template and an RPA primer according to the following steps
The Basic system is configured as follows:
for each sample, 2.5 μ L of 280mM magnesium acetate (MgOAc) was added and mixed well. If multiple samples are to be processed in this manner simultaneously, mgOAc can be added to the lid of the reaction tube (8-row tube), the tube lid carefully closed, and the reaction activated by centrifugation of MgOAc into the rehydrated material. Briefly shaken and rapidly centrifuged again.
Reaction temperature: at 37 ℃.
Reaction time: for 10min.
8. Cas13a cleavage reaction
Configuring a Cas13a reaction system by the amplification product obtained in the step 7 according to the following system:
reaction temperature: 37 ℃ is carried out.
Reaction time: and (3) 30min.
9. Measuring the fluorescence signal of the control group
A50. Mu.L reaction solution of the product in step 8 was added to a 384-well plate, and a fluorescence signal (excitation light 494nm, emission light 522 nm) was detected by a microplate reader.
10. The result of the detection
(1) A series of gradient dilutions were performed on RNA at the F-circEA cyclization sites (sample (1)) transcribed and purified in vitro, including 1uM,10nM,100pM,1pM, and 10fM. Detection was performed using the CRISPR/Cas13a fluorescence detection system of example 1. Incubation is carried out for 30 minutes at constant temperature of 37 ℃, and FAM fluorescence signals are collected on a PE full-wavelength fluorescence microplate reader. The system uses DEPC water as a negative control. As a result of detection, the system sensitivity can reach 10fM (FIG. 1).
(2) Total RNA of H1299 and 293T cells, H2228, 293T and H1299 mother cells transfected with F-circEA was extracted, respectively (sample (3)); in addition, the sample is mixed with the total RNA of the human whole blood cells by in vitro transcription target RNA for downstream detection of the target RNA (sample (2)); in addition, the target RNA in H2228 (containing the fusion gene EML4-ALK, i.e., containing F-circEA) cells was detected (sample (4)).
The results show that positive fluorescence values can be detected in the sample containing the target RNA, which indicates that the detection result of the invention is not influenced by the RNA of other samples (FIG. 2), and the specificity of the detection of the invention is high.
The beneficial effects of the present invention are demonstrated by the following experimental examples.
Experimental example 1 sequence screening and optimization of guide RNA of the present invention
5 crRNAs (the horizontal line part is the same loop structure sequence at the 5' end) on both sides of the trans-cyclization site are designed:
1：
3’-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACACUUCACACGAGACUUUUAAGCUCGUA(SEQ ID NO.7)；
2：
3’-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAAC-CUUCACACGAGACUUUUAAGCUCGUAG(SEQ ID NO.8)；
3：
3’-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCACACGAGACUUUUAAGCUCGUAGUGG(SEQ ID NO.9)；
4：
3’-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCGAGACUUUUAAGCUCGUAGUGGAAGA(SEQ ID NO.10)；
5：
3’-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGUGAUGCUCGAAUUUUCAGAGCACCACU(SEQ ID NO.1)。
the gRNA (5' -end designed with the same loop structure sequence: GAUUAGACUACCCCCAAAACGAAGGGGACUAAAC) was designed and applied to the detection system of example 1 for detection, and the detection results are shown in FIG. 3. It can be seen that, based on the best sensitivity of the 5 th crRNA sequence, which is the most preferred crRNA, the designed gRNA sequence (SEQ ID No. 1):
5 'GAUUAGACUACCCAAAACGAAGGGGACUAACGUGAUGCUCGAAUUUCAGAGCACCACU-3', named F-circEA-gRNA, is used for the kit.
In conclusion, the invention synthesizes the guide RNA with strong pertinence and high anti-interference capability through the design of the guide RNA and the fluorescent reporter molecule, can efficiently identify the lung cancer fused ring RNA and activate Cas13 to cut off the fluorescent reporter molecule, and realizes sensitive fluorescent detection; the kit for detecting F-circEA based on the guide RNA design can effectively carry out lung cancer liquid biopsy detection, and has the advantages of rapidness, low cost, low environmental use requirement and wide application prospect.
Claims (9)
1. A guide RNA is characterized in that the sequence is shown as SEQ ID NO. 1.
2. Use of the guide RNA of claim 1 in the preparation of a lung cancer detection kit.
3. A circular RNA detection kit comprising the guide RNA according to claim 1.
4. The kit of claim 3, further comprising a Cas13a protein and a fluorescent reporter molecule;
the fluorescent reporter molecule is ssRNA with two ends respectively modified with a fluorescent group and a quenching group, and the sequence is shown as SEQ ID NO. 2.
5. The kit of claim 4, wherein the fluorophore is: FAM, cy3, cy5, HEX, or ROX; the quencher group is: BHQ1, BHQ2, BHQ3 or TAMRA.
6. The kit of claim 5, wherein the fluorescent group is FAM and the quencher group is BHQ1.
7. Use of the kit of any one of claims 3 to 6 for the preparation of a lung cancer diagnostic reagent.
8. The use of claim 7, wherein the lung cancer diagnostic reagent is a reagent for detecting a biomarker of lung cancer in blood.
9. The use of claim 8, wherein the lung cancer biomarker is lung cancer fused circular RNA: F-circEA.
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