Whole genome silencer screening system and application thereof

A genome-wide, silencer technology, applied in the field of genetic engineering, can solve problems that limit the research and application of silencers

Active Publication Date: 2021-03-23
AGRI GENOMICS INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current research on non-coding sequences mainly focuses on promoters, enhancers and insulators, and there is no technology that can screen out silencers at the genome-wide level, which limits the research and application of silencers

Method used

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  • Whole genome silencer screening system and application thereof
  • Whole genome silencer screening system and application thereof
  • Whole genome silencer screening system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0025] 1. Construction of genome-wide silencer screening system (MAS-seq)

[0026] 1. Construction of silencer screening MAS-seq vector

[0027] MAS-seq vector construction process: According to the STARR-seq vector (addgene: #71509), the SCP1 promoter sequence on the STARR-seq vector was replaced with the hPGK promoter sequence, and the restriction sites Age I and Sal were deleted Between the CmR resistance gene sequence and the ccdB suicide gene sequence, the synthetic carrier is named as the MAS-seq carrier, and the main gene sequences of the carrier include: hPGK-GFP-left arm-right arm-pA site, such as figure 1 Shown, and the nucleotide sequence of the vector is shown in SEQ ID NO:1.

[0028] The constructed MAS-seq vector was used to screen silencers. Among them, silencers can not only interact with promoters and inhibit the expression of target genes, but also function independently of their positions. We inserted the sequence of the marker gene into the downstream of...

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Abstract

The invention discloses a whole genome silencer screening system. The system comprises an MAS-seq vector, wherein the MAS-seq vector comprises a strong promoter, homologous arms, a marker gene and a poly A site, and the nucleotide sequence of the MAS-seq vector is shown as SEQ ID NO: 1. When the MAS-seq vector is inserted into a silencer sub-sequence among homologous arms, the expression abundanceof marker gene mRNA can be greatly reduced, the expression quantity of the marker gene mRNA can be detected through sequencing, and the inserted sequence is indicated to be the silencer sub-sequenceif the expression quantity is low. Therefore, the system can be used for screening genomic silencers in a large scale, is sensitive and convenient to operate, and is suitable for large-scale use and wide application.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a genome-wide silencer screening system and its application. Background technique [0002] More than 98% of the human genome is non-coding sequences: including repeat regions, non-coding RNA regions, gene intron regions, etc. The DNA Encyclopedia Project (ENCODE) project has annotated these regions with epigenetic features and transcription factor binding sites, which is a very meaningful work. In recent years, it has been found that there are some important regulatory elements in these regions: such as promoters, enhancers, insulators and silencers, and these regulatory elements have been proved to play an important role in gene expression, biological phenotype and disease occurrence. However, the current research on non-coding sequences mainly focuses on promoters, enhancers and insulators, and there is no technology that can screen out silencers at the genome-wide level, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/65C12Q1/6897C12Q1/6869
CPCC12N15/63C12N15/65C12N2830/50C12Q1/6869C12Q1/6897C12Q2531/113C12Q2535/122C12Q2521/107
Inventor 张玉波朱秀生黄雷
Owner AGRI GENOMICS INST CHINESE ACADEMY OF AGRI SCI
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