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Gene knockout method

A gene knockout and gene technology, applied in the field of gene editing, can solve problems such as low integration efficiency

Inactive Publication Date: 2019-02-26
PEKING UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Repair by homologous recombination (HR) requires the construction of long flanking sequences, and the integration efficiency is low, while the integration efficiency of DNA repair by non-homologous end joining (NHEJ)[27,28] is usually higher than that of homologous Recombination repair [29]

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0165] Example 1 Enrichment of Knockout Events on ANTXR1 Gene in HeLa Cells Using Linear Donor DNA

[0166] 1. Design of sgRNA

[0167] Two sgRNAs targeting the first exon of the ANTXR1 gene in HeLa cells were designed, and their efficiency in generating deletion or insertion mutations (Indels) at the target site was verified by T7E1 assay. The verification results are shown in Table 1. where sgRNA1 ANTXR1 The targeted sequence is referred to as sg1 in this example, sgRNA2 ANTXR1 The targeted sequence is referred to as sg2 in this example.

[0168] Table 1 sgRNA targeting the first exon of ANTXR1 gene in HeLa cells

[0169]

[0170] 2. Construction of Linear Donor DNA

[0171] A total of two linear donor DNAs (Donor ANTXR1-sg2 and Donor ANTXR1-pg ), whose structure refers to figure 1 a.

[0172] Donor ANTXR1-sg2 From 5' to 3' ends include: 20bp protection sequence, sg2, reverse stop codon, CMV promoter-driven puromycin resistance gene, forward stop codon, 20bp pr...

Embodiment 2

[0182] Example 2 Enrichment of Knockout Events on the HBEGF Gene in HeLa Cells Using Linear Donor DNA

[0183] Since donors with either single- or double-cleavage sites can greatly improve the selection of cells with modifications at the target site, for convenience only single-cleavage donors were used in this example.

[0184] 1. Design of sgRNA

[0185] Two sgRNAs targeting the HBEGF gene in HeLa cells were designed, and their efficiency of generating Indels at the target site was verified by T7E1 assay. The verification results are shown in Table 3. where sgRNA1 HBEGF The target sequence against is called sg1 in this example, sgRNA2 HBEGF The targeted sequence is referred to as sg2 in this example.

[0186] Table 3 sgRNAs targeting the HBEGF gene in HeLa cells

[0187]

[0188]

[0189] 2. Construction of Linear Donor DNA

[0190] Construct a linear donor DNA (Donor HBEGF-sg1 ), whose structure refers to image 3 a.

[0191]Donor HBEGF-sg1 From 5' to 3' en...

Embodiment 3

[0197] Example 3 Enrichment of Knockout Events on HBEGF Gene in HEK293T Cells Using Linear Donor DNA

[0198] 1. Design of sgRNA and construction of linear donor DNA

[0199] Design of sgRNA2 targeting HBEGF gene in HEK293T cells HBEGF , and construct a linear donor DNA (Donor HBEGF-sg2 ), the donor includes: 20bp protection sequence from 5' to 3' end, sg2, reverse stop codon, EGFP gene driven by CMV promoter, forward stop codon, 20bp protection sequence, see Figure 4 a.

[0200] 2. Verification of gene knockout efficiency

[0201] Using a plasmid expressing Cas9 and sgRNA2 HBEGF , with or without its corresponding donor Donor HBEGF-sg2 , co-transfected HEK293T cells, cells were screened by FACS, EGFP-positive cells were screened by FACS in the group with donor, and mCherry-positive cells were screened by FACS in the group without donor. FACS-selected cells were treated with DT (40 ng / ml) to compare the effect of linear donor DNA on HBEGF knockdown efficiency. The im...

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Abstract

The invention relates to a universal donor construct, and a gene knockout method, system and kit. According to the gene knockout method in the invention, a marker gene included in the universal donorconstruct is inserted to a double-strand break position in a cell genome by non-homologous end joining, and cells in which genes are knocked out are enriched by an expressed marker, so the efficiencyof gene knockout via sequence-specific nucleases is improved. The universal donor construct of the invention comprises a universal linear donor DNA which contains a universal target sequence cleavableby the sequence-specific nucleases; the universal target sequence does not exist in the genome of a cell to undergo gene knockout; therefore, when performing gene knockout, it is not necessary to specifically construct a matching linear donor DNA, and a general linear donor DNA and a gRNA targeting to the universal linear donor DNA can be directly used.

Description

technical field [0001] The invention relates to gene editing technology, in particular to a gene knockout method. Background technique [0002] Gene editing technology has revolutionized the experimental study of gene function. The three main technologies, ZFNs (zinc finger nucleases) [1], TALENs (transcription activator-like effector nucleases) [2-4] and the CRISPR / Cas9 system [5-7], use different mechanisms to generate sequences Specific double-strand breaks (DSBs) and subsequent triggering of natural repair systems to complete sequence-specific modifications [8,9]. These techniques are widely used in functional gene research [10], dynamic and real-time imaging of chromosomal loci [11,12], correction of disease mutations [13], gene therapy [14], etc. The CRISPR / Cas9 system has become particularly popular due to its high efficiency and ease of operation. The CRISPR / Cas9 system was originally used by the bacterial immune system to resist foreign viruses or plasmids. In th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/90
CPCC12N15/113C12N15/902C12N2310/10
Inventor 魏文胜陈一欧周悦欣张鸿敏袁鹏飞刘源
Owner PEKING UNIV
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