Mouse epigenetic gene knockout screening library and its construction method

Abstract

The invention relates to a mouse epigenetic gene knockout screening library and a construction method thereof. The mouse epigenetic gene knockout screening library includes an SgRNA expression plasmid targeting epigenetic related genes, and the targeting epigenetic related genes includes Amino acid acetyltransferase gene, histone deacetylase gene, histone lysine methyltransferase gene, histone lysine demethylase gene, protein arginine methyltransferase gene, histone recognition , DNA methylation-related genes, RNA methylation-related genes, chromatin remodeling complex genes, chromatin assembly and histone chaperone genes. The gene library constructed by the present invention is a dual-plasmid expression system, the plasmid is relatively small, and the construction is relatively easy to operate; at the same time, the screening library is only for epigenetic genes, the library is relatively small, and data analysis is relatively easy to perform single-cell sequencing.

Description

technical field [0001] The invention relates to a mouse epigenetic gene knockout screening library and a construction method thereof, and belongs to the field of gene biotechnology. Background technique [0002] CRISPR / Cas9 gene knockout technology is currently the most widely used and most efficient gene editing technology, and a variety of gene editing methods have been developed on the basis of this technology, including single-base mutation, homologous recombination based on CRISPR / Cas9 technology, etc. The method has been widely used in gene therapy, drug screening and other fields. [0003] The basic principle of CRISPR / Cas9 is that under the targeting of sgRNA, the Cas9 protein can recognize and bind to the editing position of the target gene, cut the genome to cause double-stranded DNA breaks, and cause DNA damage repair in cells. The main DNA damage repair pathway in cells is Non-homologous End Joining (NHEJ), which can lead to insertion or deletion (Indel) in the ...

AI Technical Summary

Problems solved by technology

The main DNA damage repair pathway in cells is Non-homologous End Joining (NHEJ), which will lead to insertion or deletion (Indel) of the target gene genome, making the target gene unable to function normally. Transcription and translation, resulting in gene knockout

[0004] The deficiencies of the existing knockout screening libraries are as follows: 1. Knockout libraries for the entire genome genes, including human and mouse genome-wide knockout libraries, have not been designed to specifically target epigenetic factors. In addition to the library; 2. It cannot be well combined with single-cell sequencing technology, and multiple single-cell sequencing is required to obtain the knockout sequencing results of the entire library; 3. The current knockout screening library is a mixed library of all knockout plasmids , the amplification efficiency of each gene knockout plasmid is different during the amplification process, which leads to the unobjective results of subsequent knockout screening; 4. The current knockout screening library is a single-plasmid expression system, compared to the double-plasmid system , the library construction of the single-plasmid system is more complicated in the library construction process

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