CRISPR-Cas9-based nucleic acid detection system and application thereof

A detection system and nucleic acid technology, applied in the fields of synthetic biology and biomedicine, can solve problems such as low reprogramming efficiency

Active Publication Date: 2020-02-11
SUZHOU DIYINAN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] However, the efficiency of dCas9-based transcriptional regulation and reprogramming is currently low. Single-stranded guide RNA (sgRNA) is used to regulate gene expression. The design is simpler, flexible and efficient, and it can also avoid the influence of fusion proteins on Cas9 function.

Method used

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  • CRISPR-Cas9-based nucleic acid detection system and application thereof
  • CRISPR-Cas9-based nucleic acid detection system and application thereof
  • CRISPR-Cas9-based nucleic acid detection system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1 Design standard sgRNA and RsgRNA

[0079] Standard sgRNA targets were designed by CHOPCHOP (https: / / chopchop.rc.fas.harvard.edu / ), and screened and evaluated by Moreno-Mateos scores (Vejnar C E, et al..Cold Spring Harbor Protocols, 2016), obtained as The sgRNA shown in SEQ ID NO:2, the secondary structure is as follows figure 1 Shown; The RsgRNA shown in SEQ ID NO:3 increases the antisense nucleic acid shown in SEQ ID NO:1 at the 5' end of sgRNA, and the secondary structure is as shown in figure 2 shown.

[0080] Such as image 3 As shown, in the presence of the target nucleic acid, the trigger sequence hybridizes with the antisense nucleic acid on the RsgRNA, and the free energy of the secondary structure ΔG=-57.68kcal / mol.

Embodiment 2

[0081] Construction of embodiment 2 reporter plasmid

[0082] The reporter plasmid constructed in this example includes T7 promoter, ribosome binding site, G6PD coding sequence and T7 terminator. According to the protein sequence of G6PD, combined with the characteristics of E. coli codon optimization, the design is shown in SEQ ID NO: 4 The G6PD coding sequence, the steps are as follows:

[0083] Using the Hanbio HB-infusionTM seamless cloning kit, using the DNA exonuclease, DNA polymerase and ligase in the system to insert the coding sequence shown in SEQ ID NO: 4 into the vector pET3a backbone, avoiding restriction internal Dicer cloning method on the impact of the promoter region and ribosome binding site sequence Shine-Dalgarno (SD), constructed as Figure 4 Plasmids indicated.

Embodiment 3

[0084] Example 3 Regulatory effect of Cas9-sgRNA or Cas9-RsgRNA on G6PD

[0085] The Cas9-sgRNA or Cas9-RsgRNA constructed in Example 1 was added to the cell-free protein expression system containing the reporter plasmid to regulate the synthesis of G6PD protein.

[0086] Such as Figure 5 Indicates the cleavage effect of Cas9-RsgRNA on DNA and the inhibitory effect of different concentrations of target substances on the cleavage substrate. Among them, the leftmost lane is the DNA ladder lane, lane 1 indicates DNA as the cleavage substrate, and lane 2 indicates Cas9-RsgRNA The cleavage products after cleavage of substrate DNA, lanes 3-8 represent the inhibitory effect of different concentrations of trigger sequences (0.1, 0.4, 2, 10, 50, 250nM) on the Cas9-RsgRNA cleavage system. The results showed that with the increase of the concentration of the trigger sequence, its inhibitory effect on Cas9-RsgRNA cleavage was stronger.

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Abstract

The invention provides a CRISPR-Cas9-based nucleic acid detection system and application thereof. A protein expression regulating and control system comprises Cas9 protein, RsgRNA and a cell-free protein expression system; and the RsgRNA comprises antisense nucleic acid and sgRNA according to a direction of 5' to 3'. The nucleic acid detection system provided by the invention combines a CRIPSR/Cas9 nucleic acid identification regulating and control system with a gene loop, regulates and controls the expression of protease by the RsgRNA, converts a nucleic acid signal into generation of the protease, and realizes rapid detection of the nucleic acid by detecting the protease.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and the technical field of synthetic biology, and relates to a nucleic acid detection system based on CRISPR-Cas9 and its application. Background technique [0002] Nucleic acid detection technology is of great significance to the protection of people's life and health, and has great economic value. Nucleic acid detection provides a safe, rapid, sensitive and specific means for disease diagnosis and food supervision. The current methods for nucleic acid detection mainly include electrophoresis, spectroscopy and fluorescence methods, but these methods are time-consuming, laborious and costly. [0003] Synthetic biology aims to solve global challenges by designing components of natural systems. Currently, synthetic biologists are building whole-cell biosensors, the core of which is to prepare synthetic gene networks that can be artificially controlled. But housing these engineered pathways in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/113C12N15/90C12Q1/37
CPCC12N15/63C12N15/113C12N15/902C12Q1/37C12N2310/20
Inventor 徐峰张超张鹏晖
Owner SUZHOU DIYINAN BIOTECHNOLOGY CO LTD
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