Biosensor and kit based on CRISPR/Cas12a system, and application of biosensor and kit in small molecule detection

A technology of small molecules and kits, applied in the field of in vitro detection of small molecules, can solve problems such as undetected small molecule compounds

Active Publication Date: 2020-10-23
EAST CHINA UNIV OF SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still no reports on the use of CRISPR / Cas systems for the detection of small molecule compounds

Method used

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  • Biosensor and kit based on CRISPR/Cas12a system, and application of biosensor and kit in small molecule detection
  • Biosensor and kit based on CRISPR/Cas12a system, and application of biosensor and kit in small molecule detection
  • Biosensor and kit based on CRISPR/Cas12a system, and application of biosensor and kit in small molecule detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0224] Embodiment 1 Uric acid biosensor

[0225] In this example, the allosteric transcription factor HucR and the activated dsDNA fragment (dsDNA hucR ; Wherein, comprising HucR binding site (with the gRNA complementary sequence contained therein) and CRISPR / Cas12a recognition site (PAM)) as the recognition element, with CRISPR / Cas12a, gRNA and single-stranded DNA (ssDNA; both ends marked with FAM fluorophore and BHQ fluorescence quencher (as a probe) are used as transducing elements to construct a biosensor platform capable of sensing uric acid.

[0226] HucR protein is a repressor protein derived from Deinococcus radiodurans, and its amino acid sequence can be shown in SEQ ID NO:1. Such as figure 1 As shown, in the absence of uric acid (UA), HucR can bind dsDNA hucR HucR interaction site on HucR-dsDNA hucR Complex. When uric acid is present, uric acid specifically binds HucR, making dsDNA hucR Release of episomal dsDNA from HucR hucR , dsDNA in episomal form hucR A...

Embodiment 2 4

[0255] Example 2 Tetracycline biosensor and p-hydroxybenzoic acid (p-HBA) sensor

[0256] In order to verify the universality of the sensor and method of the present invention, adopting the concept of Example 1, sensors responsive to tetracycline and p-HBA were designed respectively. Tetracycline-responsive biosensors utilize the allosteric transcription factor TetR and double-stranded activating DNA fragments (dsDNA TetR ; which contains TetR binding site and CRISPR / Cas12a recognition site (PAM)) as recognition elements, with CRISPR / Cas12a, gRNA and single-stranded DNA (ssDNA; both ends are labeled with FAM fluorophore and BHQ fluorescence quencher, as Probe) as the transducer element. A p-HBA-responsive biosensor utilizes the allosteric transcription factor HosA and a double-stranded activating DNA fragment (dsDNA HosA ; which contains HosA binding site and CRISPR / Cas12a recognition site (PAM)) as recognition elements, with CRISPR / Cas12a, gRNA and single-stranded DNA (ssDN...

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Abstract

The invention relates to a biosensor, composition and kit comprising an allosteric transcription factor and a CRISPR/Cas12a system, and related methods and application thereof. The mechanism of the biosensor is that a small molecule detection tool mediated by the allosteric transcription factor and the CRISPR/Cas12a is adopted; through combination of a target small molecule and the allosteric transcription factor, affinity between the allosteric transcription factor and double-stranded DNA is changed; and cis-cleavage activity and/or trans-single-stranded DNA cleavage activity of CRISPR/Cas12aare/is adopted for cutting a dsDNA probe and/or a ssDNA probe so as to convert a target small molecule signal into a corresponding optical signal. With the biosensor, the composition, the kit and therelated methods in the invention, time and cost can be saved when small molecules are detected in vitro, sensitivity is high, and the potential of high-throughput detection is achieved.

Description

technical field [0001] The invention belongs to the field of biological detection, and relates to a biological system that converts small molecular signals into light signals through the cis-cleavage and trans-cleavage activities of CRISPR / Cas12a by means of an allosteric transcription factor (aTF) and a CRISPR / Cas12a system. Sensors, compositions and kits, and methods for detecting small molecules in vitro using the same. Background technique [0002] The reason why small molecule detection is so important is mainly reflected in the application in various fields such as clinical diagnosis, drug development, food safety and environmental monitoring. For example, as the end product of purine metabolism, uric acid is generally produced and excreted in a dynamic balance in the body, and excessive accumulation will affect the normal function of human cells. Among them, hyperuricemia and gout and kidney disease all use uric acid as an indicator, which is a biomarker for detectio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N1/34G01N1/28
CPCG01N21/6486G01N1/34G01N1/28G01N21/64C12Q1/6876Y02A50/30
Inventor 张立新谭高翼梁敏东王为善刘家坤
Owner EAST CHINA UNIV OF SCI & TECH
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