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Method for detecting cell endogenous low-abundance gene and lncRNA level

An endogenous, cellular technology that can be used in biochemical equipment and methods, DNA/RNA fragments, genetic engineering, etc., to solve problems such as low expression levels and untranslatability

Pending Publication Date: 2022-06-07
CENT FOR EXCELLENCE IN BRAIN SCI & INTELLIGENCE TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, functional annotation of lncRNAs is extremely challenging because they are not translatable and they are usually expressed at lower levels compared to coding RNAs

Method used

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  • Method for detecting cell endogenous low-abundance gene and lncRNA level
  • Method for detecting cell endogenous low-abundance gene and lncRNA level
  • Method for detecting cell endogenous low-abundance gene and lncRNA level

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0180] Example 1: Development of the SPH-OminiCMV-Ents system

[0181] The key design of the ent-switch is to target the insertion of the sgRNA precursor into the transcribed region of the endogenous gene. The sgRNA precursor is flanked by sequences released after transcription that can be recognized by endogenous mechanisms, such as figure 1 a shown. Targeted genes and sgRNA precursors are transcribed into single transcripts driven by endogenous promoters, and then mature sgRNAs are released through an endogenous cleavage program, such as figure 1 a shown.

[0182] In this example, a highly sensitive CRISPR activator-related reporter system is provided to efficiently detect the presence of released sgRNAs. In this system, mCherry expression was induced by targeting a minimal promoter (mini-promoter) and using the CRISPR-activator Suntag-P65-HSF1 (SPH) activation system previously developed by the inventors.

[0183] In order to induce effective activation, sgRNAs were pre...

Embodiment 2

[0201] Example 2: SPH-OminiCMV-Ents can amplify the signals of low-abundance genes and track the dynamic expression of genes during differentiation

[0202] Fluorescence visualization techniques provide an easy and direct way to capture spatiotemporal information of cellular events. To explore whether SPH-OminiCMV-Ents can faithfully reflect the expression levels of target genes, tRNA-sgRNA-tRNAs were inserted into the 3'UTR regions of eight differentially expressed genes, including a highly expressed housekeeping gene Actb and seven pluripotent genes. sex-related genes ( figure 2 a).

[0203] The results showed that the gene expression level was significantly correlated with mCherry intensity ( figure 2 b, c, r2=0.8644, p=0.0008), indicating that SPH-OminiCMV-Ents can be used as a reporter system to monitor gene expression levels at the cellular level.

[0204] The fluorescence intensity induced by the SPH-OminiCMV-Ent and P2A-mCherry strategies was also compared in this e...

Embodiment 3

[0212] Example 3: Detection of lncRNA activity using SPH-OminiCMV-Ents

[0213] Long noncoding RNAs (lncRNAs) have been identified to play important roles in a variety of biological processes, and their expression is often restricted to specific cell types or at specific developmental stages. However, detection and functional analysis of lncRNAs is very difficult because conventional strategies for co-expression of fluorescent proteins are not applicable to non-translated genetic elements. LncRNAs are also difficult to study because they are often expressed at very low levels.

[0214] In this example, it was tested whether SPH-OminiCMV-Ents could be used to detect lncRNAs. The tRNA-sgRNA-tRNA cassettes targeting mCherry were inserted into the 3'UTRs of two lncRNAs, malt1 and Lncenc1, respectively, on SPH-OminiCMV mESC cell lines. Interestingly, the results showed that mCherry was expressed in SPH-OminiCMV-Ents-Malat1 and SPH-OminiCMV-Ents-Lncenc1 cells, but not in SPH-Omini...

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Abstract

The invention provides a method. Specifically, the invention provides the sgRNA response type promoter, a detection system which contains the sgRNA response type promoter and is used for detecting the endogenous nucleic acid of the cell, and a corresponding detection method. The endogenous gene with very low expression level in the cell can be reliably detected by using the detection system or method provided by the invention; and the transcriptional activity of long non-coding RNA (lncRNA), which is usually low in expression, can also be monitored in living cells. In addition, the method can amplify endogenous signals to visualize the dynamic process of expression of low-abundance genes and lncRNA in cells, and a powerful platform is provided for detecting the activity of endogenous genetic elements and potential cell functions of the endogenous genetic elements.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for detecting the level of endogenous low-abundance genes and lncRNA in cells. Background technique [0002] Detection of endogenous signals and precise control of gene pathways in the natural environment, as well as precise manipulation of genetic information, are critical for understanding biological processes as well as identifying fundamental biological design principles of organisms and enabling higher levels of regulation in diverse systems. [0003] Previous research has developed a series of genetic switches that act as sensors or perform other functions. However, these strategies are mainly limited by the ability to process endogenous signals. Single guide RNA (sgRNA), similar to a GPS, can direct Cas nucleases to target genomic loci with specificity, efficiency and versatility. Although various inducible sgRNAs have been developed to receive endogenou...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N9/22G01N21/64
CPCC12N15/113C12N9/22G01N21/6428C12N2310/20
Inventor 杨辉周海波高妮
Owner CENT FOR EXCELLENCE IN BRAIN SCI & INTELLIGENCE TECH CHINESE ACAD OF SCI