Method for constructing eukaryon gene knockout library by using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system

Abstract

The invention provides a method for constructing a eukaryon gene knockout library by using a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas9 system. The method comprises the following steps: expressing genes encoded with Cas9 and OCT1 (Organic Cation Transporter 1) proteins into a eukaryon cell line; screening to obtain a cell line which stably expresses Cas9; and carrying out library construction and functional screening. The method has the greatest advantages that the method can be applied to most of eukaryon cell lines and is not limited by the specific cell line. Furthermore, the functional screening positive rate is high and the background value is low. According to a large-scale screening method, the cost is greatly reduced; the problems that the time is long and the labor cost is high due to the fact that a single gene knockout cell is prepared are solved.

Description

technical field [0001] The invention relates to the fields of genomics and genetic engineering, in particular to a method for constructing a eukaryotic gene knockout library using a CRISPR / Cas9 system. Background technique [0002] Gene knockout is a technical means to target a specific gene by destroying or changing its gene sequence to lose its function. Commonly used gene knockout methods include: zinc finger nucleases (ZFNs) (Miller et al., 2007; Porteus and Baltimore, 2003; Wood et al., 2011), transcription factor activator-like nuclease (TALEN) (Miller et al. al.,2011;Wood et al.,2011;Zhang et al.,2011), and the recently discovered second type of adaptive immune system CRISPER / Cas9 system in prokaryotes (Cong et al.,2013;Mali et al., 2013) etc. The CRISPER / Cas9 system was originally used by the bacterial immune system to defend against foreign viruses or plasmids. In the second type of CRISPER system, Cas9 endonuclease cuts double-stranded DNA under the guidance of ...

AI Technical Summary

Problems solved by technology

[0004] However, shRNA libraries are not gene knockout libraries and can only partially silence gene expression

There are many disadvantages, such as the effect on the expression of specific genes often cannot lead to phenotypic changes; the expression of irrelevant genes may be suppressed by mistake, etc.

Although some gene knockout libraries have been reported, especially KBM7 cells, which are partially haploid cells, can be used to construct knockout libraries, but there are problems with their stability and efficiency, and the system is limited to specific cell lines, limited range of applications

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