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Cas12a-CcrRNA system based on regulation and control of nuclease and circular guide RNA and application of Cas12a-CcrRNA system

A nucleic acid and ribozyme technology, applied in the field of analysis and detection, to achieve the effects of high sensitivity, strong anti-interference and good specificity

Pending Publication Date: 2022-03-25
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of nucleases for circular guide RNA ( C crRNA) cleavage to release L There are no reports on the crRNA-activated Cas12a system, which is of great significance for expanding the detection of non-nucleic acid targets based on CRISPR-Cas

Method used

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  • Cas12a-CcrRNA system based on regulation and control of nuclease and circular guide RNA and application of Cas12a-CcrRNA system
  • Cas12a-CcrRNA system based on regulation and control of nuclease and circular guide RNA and application of Cas12a-CcrRNA system
  • Cas12a-CcrRNA system based on regulation and control of nuclease and circular guide RNA and application of Cas12a-CcrRNA system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Based on the Cas12a system regulated by nuclease and circular guide RNA and constructing the technical route of the biosensor

[0050] This application is based on the Cas12a system regulated by nuclease and circular guide RNA and the technical route of constructing a biosensor mainly includes the following steps: (1) C Validate the feasibility of crRNA regulation on the Cas12a system, explore C Effect of crRNA on cis-cleavage and trans-cleavage kinetics of Cas12a protein; (2) Characterization of Cas12a-crRNA binding affinity; (3) Construction of Cas12a system based on nuclease activation; (4) Construction of ATP and two pathogenic bacteria To explore the sensitivity and selectivity of the sensor for target detection, and to use it in the diagnosis of actual clinical samples, to explore the sensitivity and specificity for clinical diagnosis.

Embodiment 2

[0051] Example 2 Exploration C Regulation of the function of Cas12a by crRNA

[0052] The present invention builds based on nuclease and C The schematic diagram of the crRNA-regulated Cas12a system is as follows: figure 1 shown, is based on C crRNA can reduce the cleavage activity of Cas12a to DNA, so the present invention explores at first C The regulatory effect of crRNA on the function of Cas12a mainly includes C The effect of crRNA on the reaction kinetics of Cas12a cis-cutting dsDNA and trans-cutting ssDNA.

[0053] (1) C crRNA ii Preparation: 5' phosphorylation L crRNA (such as L crRNA i , L crRNA ii , 100pmol) first with the ligation template (such as LT i , LT ii , 110pmol) and 10μL 10×T4 RNA Ligase2 (T4RL2) buffer (500mM Tris hydrochloric acid, 20mM magnesium chloride, 10mM DTT, 4mM ATP) were mixed, heated to 90°C for 2 minutes, then cooled at room temperature (RT) for 15 minutes; Add 50U of T4RL2, 100U of RNase inhibitor and nuclease-free water to the m...

Embodiment 3

[0058] Example 3 C Affinity characterization of crRNA and Cas12a

[0059] (1) Dot blot experiment

[0060] A circular test area with a diameter of 4mm was printed on a nitrocellulose membrane (Millipore HF120) by a wax jet printer (Xerox ColourQube 8570N), and heated at 120°C for 2min to melt the wax into the membrane to form a hydrophobic barrier. 1 μL of Cas12a (148ng / uL) was spotted on each test area. After drying at room temperature for 10 minutes, block with 10 μL of 1×BB solution containing 1% BSA for 30 minutes. After washing twice with 20 μL 1×WB, add 10 μL 100 nM FAM-labeled L crRNA ii or C crRNA ii , and incubate for 30 minutes. Place the cardboard in a tank of 2 mL 1×WB to wash for 5 minutes, and after drying, use a fluorescence imager to scan and image.

[0061] Qualitative analysis by dot blot experiments, image 3 a display L crRNA ii The binding affinity to Cas12a is significantly better, while the C crRNA ii have poor binding affinity.

[0062] (2...

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Abstract

The invention discloses a Cas12a-CcrRNA system based on regulation and control of nuclease and circular guide RNA and application of the Cas12a-CcrRNA system, and belongs to the technical field of analysis and detection. It is found for the first time that CcrRNA synthesized through an enzymatic ligation reaction can reduce the activity of Cas12a on double-stranded DNA specific cleavage (cis) and single-stranded DNA non-specific cleavage (trans), when nuclease (NAzyme) cleaves CcrRNA, released LcrRNA can recover the activity of Cas12a, a nuclease activated Cas12-CcrRNA system (NA3C) is constructed, and the CcrRNA can be used as a Cas12-CcrRNA system (NA3C), so that the CcrRNA can be used for detecting the activity of Cas12a. The invention further designs a high-specificity detection method for ATP (adenosine triphosphate) and pathogenic bacteria (escherichia coli and klebsiella pneumoniae), and the detection limits of the high-specificity detection method are as low as 500nM and 102CFU / mL respectively; the constructed NA3C system is further used for clinical evaluation of escherichia coli, and the result shows that the diagnosis sensitivity can reach 100%, the specificity is 90%, and the NA3C has the potential of being used for clinical diagnosis.

Description

technical field [0001] The invention belongs to the technical field of analysis and detection, and in particular relates to a Cas12a- C crRNA system and its application in biosensing. Background technique [0002] Molecular biosensing is crucial for clinical diagnosis, biosafety, food safety, and environmental monitoring. At present, CRISPR-Cas system has been widely used in the field of biosensing as a signal amplification tool. Researchers have established many nucleic acid detection methods based on the CRISPR-Cas system, which are widely used in the diagnosis of infectious or non-infectious diseases. However, the number of sensing methods for non-nucleic acid biomarkers (such as small molecules, proteins, and metal ions) is very limited and urgently needs to be expanded. [0003] In nature, covalently closed circular RNAs (circRNAs) widely exist in viroids, mimiviruses, and spliced ​​introns or exons in eukaryotes, bacteria, and archaea. CircRNAs, which can be prepar...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N9/22C12Q1/10G01N21/64
CPCC12N15/113C12N9/22C12Q1/10G01N21/6486C12N2310/20Y02A50/30
Inventor 刘猛吴云萍常洋洋
Owner DALIAN UNIV OF TECH
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