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

A gene knockout and eukaryotic technology, applied in the fields of genomics and genetic engineering, can solve problems such as limited application range, system limitations, and phenotypic changes, and achieve cost reduction, low background value, and high positive rate Effect

Active Publication Date: 2014-03-26
EDIGENE BIOTECH INC
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  • Abstract
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  • Application Information

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, et

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Construction of CRISPER / Cas9 gene knockout library and screening of genes related to anthrax toxin cytotoxicity

[0031] 1. Design of library sgRNA

[0032] For 296 genes, 2-3 sgRNA target sites were found for each gene, see Table 1 for details.

[0033] Table 1 Gene composition of sgRNA library, sgRNA target region and primer sequence used to construct sgRNA plasmid

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[0062] 2. Screening of HeLa cells highly expressing Cas9

[0063] (1) The DNA sequences of OCT1 and Cas9 were connected by 2A and loaded onto the lentiviral vector pLenti-CMV-MCSSV-Bsd by the Gibson cloning method;

[0064] (2) Transfer the above-mentioned vectors i...

Embodiment 2

[0085] Example 2 Construction of CRISPER / Cas9 gene knockout library and screening of genes related to diphtheria toxin cytotoxicity

[0086] 1. Design of library sgRNA

[0087] For 296 genes, 2-3 sgRNA target sites were found for each gene, see Table 1 for details.

[0088] 2. Screening of HeLa cells highly expressing Cas9

[0089] (1) The DNA sequences of OCT1 and Cas9 were connected by 2A and loaded onto the lentiviral vector pLenti-CMV-MCSSV-Bsd by the Gibson cloning method;

[0090] (2) Transfer the above-mentioned vectors into the target cells by packaging lentivirus infection;

[0091] (3) Add blasticidin to the culture medium of the above cells for screening, and select a single clone stably expressing Cas9;

[0092] 3. Construction of Library Plasmids

[0093] (4) Look for the sgRNA action site, the sequence is 5'-G-Nx-NGG-3', where 19≤x≤22;

[0094] (5) Synthesize sgRNA monomers targeting the above-mentioned action sites. For each site, the sequence is, forward: ...

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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 ...

Claims

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Application Information

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IPC IPC(8): C40B50/06C40B40/06C12Q1/68
CPCC12N15/102
Inventor 魏文胜周悦欣朱诗优蔡昌祖
Owner EDIGENE BIOTECH INC
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