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PCr-NHEJ (non-homologous end joining) carrier as well as construction method of pCr-NHEJ carrier and application of pCr-NHEJ carrier in site-specific knockout of bacterial genes

A construction method and a technology for targeted knockout, applied in the field of genetic engineering, can solve the problems of cumbersome operation, difficult operation, and insufficient gene knockout efficiency, and achieve the effects of low operation difficulty, reduced operation difficulty, and high gene knockout efficiency.

Inactive Publication Date: 2015-06-03
GUANGDONG MEDICAL UNIV
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  • Summary
  • Abstract
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AI Technical Summary

Problems solved by technology

Therefore, the CRISPR-Cas9 system of the prior art has the disadvantages of cumbersome operation, difficult operation, and insufficient gene knockout efficiency.

Method used

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  • PCr-NHEJ (non-homologous end joining) carrier as well as construction method of pCr-NHEJ carrier and application of pCr-NHEJ carrier in site-specific knockout of bacterial genes
  • PCr-NHEJ (non-homologous end joining) carrier as well as construction method of pCr-NHEJ carrier and application of pCr-NHEJ carrier in site-specific knockout of bacterial genes
  • PCr-NHEJ (non-homologous end joining) carrier as well as construction method of pCr-NHEJ carrier and application of pCr-NHEJ carrier in site-specific knockout of bacterial genes

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Effect test

Embodiment 1

[0061] A pCr-NHEJ vector, the sequence of the pCr-NHEJ vector has the sequence shown in SEQ ID NO:1. The pCr-NHEJ vector of this embodiment can be used for bacterial gene knockout, and has the advantage of high gene knockout efficiency.

Embodiment 2

[0063] The application of a pCr-NHEJ vector of Example 1 in site-specific knockout of bacterial genes.

Embodiment 3

[0065] The construction method of a pCr-NHEJ vector of Example 1, includes the following steps:

[0066] (1) Use broad host plasmid of gram-negative bacteria P BBR1MCS-2 (GI: 773412) as the basic carrier; cas9

[0067] (2) The following three protein genes were amplified using the upstream primer and downstream primer with the prokaryotic promoter PLtetO-1 for mRNA transcription: cas9 (GI:674296984), ku (GI:444893469) and ligD (GI:444893469), respectively cas9 Amplification products, ku Amplification products and ligD Amplification product; among them, cas9 , ku with ligD The open reading frames of the three protein genes are all located downstream of independent PLtetO-1, so that cas9 , ku with ligD The expression of the three protein genes is regulated by the promoter; cas9 Amplification products, ku Amplification products and ligD There is a 15nt base overlap between the amplified products;

[0068] (3) cas9 The 3’ end of the amplified product and ku The 5'end of the amplified...

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Abstract

The invention belongs to the technical field of gene engineering, in particular to a pCr-NHEJ (non-homologous end joining) carrier as well as a construction method of the pCr-NHEJ carrier and an application of the pCr-NHEJ carrier in site-specific knockout of bacterial genes. The sequence of the pCr-NHEJ carrier is shown as the SEQ ID NO.1. The application of the pCr-NHEJ carrier in site-specific knockout of the bacterial genes is provided. The technical principle is as follows: NHEJ and CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR associated protein 9) are jointly applied, so that after DNA (deoxyribonucleic acid) is broken when a CRISPR-Cas9 system cuts bacterial genomic DNA, the NHEJ system can be connected with a broken DNA end automatically, bacteria are survived, double-strand DNA having homology with a target sequence is not required to be introduced artificially to repair the broken DNA end, and the operation steps of the CRISPR-Cas9 technique are simplified.

Description

technical field [0001] [The present invention relates to the technical field of genetic engineering, in particular to a pCr-NHEJ vector and its construction method and its application for bacterial gene knockout. Background technique [0002] "Science" published in 2013 reported a simple and efficient gene-directed editing technology - CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat; CRISPR-associated, CRISPR-Cas9). The technology system consists of a Cas9 nuclease from Streptococcus, a sequence-constant tracrRNA (trans-activating crRNA) and a 20nt-crRNA (CRISPR RNAs) specifically complementary to the target gene. The advanced structure formed by the 3' end of tracrRNA binds and activates Cas9, the 5' end of tracrRNA is complementary to the 3' end of crRNA, and the 5' end of crRNA is complementary to the target gene, thereby guiding Cas9 to reach and cut off the target sequence. Now two small RNAs have been fused into one RNA chain, referred to as sgRN...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/66C12N1/21
Inventor 赵祖国喻云梅米娜刘仿李国明
Owner GUANGDONG MEDICAL UNIV
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