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Bacillussubtilis CRISPR-Cas9 genome editing system

A Bacillus subtilis genome editing technology, applied in the field of genome editing, can solve the problems of low accuracy, low editing efficiency, and unrealized iterative cycle system, and achieve high accuracy and realize the effect of iterative editing cycle

Active Publication Date: 2019-12-31
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional scarless genome editing system in Bacillus subtilis mainly relies on the "negative selection" method, which not only has low editing efficiency and accuracy, but also cannot meet the needs of multi-site modification
In recent years, there have been reports on the editing of microbial genomes using CRISPR systems, but at present, in the reports on CRISPR gene editing of Bacillus subtilis, a streamlined iterative cycle system has not been realized, and simultaneous editing of multiple sites has not been realized.

Method used

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  • Bacillussubtilis CRISPR-Cas9 genome editing system
  • Bacillussubtilis CRISPR-Cas9 genome editing system
  • Bacillussubtilis CRISPR-Cas9 genome editing system

Examples

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Effect test

Embodiment 1

[0047] Example 1 Construction of tool plasmids pBSCas9, pBSCas9n, pDonor and pHG

[0048] The pBSCas9, pBSCas9n and pDonor tool plasmids are used in the operation process of the present invention, and the specific construction method is as follows.

[0049] (1) Construction of tool plasmid pBSCas9 and pBSCas9n

[0050] Using B. subtilis 168 genome as template, using primers P43-F and P43-L to amplify the P43 promoter sequence (SEQID NO.31), using plasmid pUC18 as template, using primers ColE1-F and ColE1-L to amplify pUC18 replication Sub-ColE1 sequence, using primers P43-F and ColE1-L to fusion the above two fragments into fragment F1. In this step of fusion, the sequence of gRNA binding to Cas9 protein and terminator sequence are introduced downstream of the P43 promoter through primers. Using plasmid pHP13 as a template, using primers Cm-F and Cm-L to amplify the chloramphenicol resistance gene and its promoter sequence (Cm), using plasmid pEBs-cop1 as template, using primers re...

Embodiment 2

[0065] Example 2: Principle of CRISPR-Cas9 system gene editing

[0066] The principle of CRISPR-Cas9 system gene editing is as follows Figure 4 Shown.

[0067] (1) Principle of unit point editing

[0068] After determining the genome modification site, select the appropriate PAM site in the region to be modified, and determine the gRNA sequence (the 20bp sequence upstream of the PAM site is the gRNA sequence). The gRNA sequence was inserted into the downstream region of the P43 promoter of the pBSCas9 plasmid. The specific method is to design primers to perform full-plasmid amplification PCR on plasmid pBSCas9, introduce BsaI restriction sites and a part of gRNA sequence into the upstream and downstream primers, and reconnect them into circular plasmids through the Golden Gate cloning reaction. In this process, insert gRNA into P43 Downstream of the promoter (SEQ ID NO. 31), this process does not introduce any extra bases.

[0069] According to the PAM site and the type of gene edi...

Embodiment 3

[0077] Example 3: Principles of CRISPR-Cas9n system genome editing

[0078] The principle of CRISPR-Cas9n system gene editing is as follows Figure 4 Shown.

[0079] (1) Principle of unit point editing

[0080] After determining the genome modification site, select the appropriate PAM site in the region to be modified, and determine the gRNA sequence (the 20bp sequence upstream of the PAM site is the gRNA sequence). The gRNA sequence was inserted into the downstream region of the P43 promoter of the pBSCas9n plasmid. The specific method is to design primers to perform full-plasmid amplification PCR on plasmid pBSCas9n, introduce BsaI restriction sites and a part of gRNA sequence into the upstream and downstream primers, and reconnect them into circular plasmids through the Golden Gate cloning reaction. In this process, insert gRNA into P43 Downstream of the promoter (SEQ ID NO. 31), this process does not introduce any extra bases.

[0081] According to the PAM site and the type of g...

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Abstract

The invention discloses a bacillussubtilis CRISPR-Cas9 genome editing system. A building method of the system comprises the following steps of (1) building a plasmid pBSCas9; (2) building a plasmid pDonor; (3) building an auxiliary plasmid pHG in a multi-site gRNA expression process; and (4) forming the bacillussubtilis CRISPR-Cas9 genome editing system by the pBSCas9, the pDonor and the pHG. Thebacillussubtilis CRISPR-Cas9 genome editing system solves the problems in the existing bacillussubtilis conventional genome editing technology; no negative selection marker is introduced; and a CRISPR-Cas9 and CRISPR-Cas9n genome non-trace editing system with high efficiency and high precision is built. The single-site and multi-site different-type gene editing operation can be realized; and meanwhile, the iterative editing cycle of an editing flow process can be realized.

Description

Technical field [0001] The invention belongs to the technical field of genome editing, and specifically relates to a Bacillus subtilis CRISPR-Cas9 and CRISPR-Cas9n genome editing system and a construction method and application. Background technique [0002] Bacillus subtilis (Bacillus subtilis) is a dominant microbial species that exists in soil and plant micro-ecosystems, and it exists widely in nature. It is non-pathogenic, harmless to humans and animals, and does not pollute the environment. It has strong resistance to stress and antibacterial and disease prevention effects. It is widely used as a model organism for molecular biology research and as a host for industrial production of high-yielding strains. [0003] With the development of systems biology and the need for the development of microbial cell factories, efficient genome editing technology has become a research hotspot in the study of metabolic network regulation. The traditional traceless genome editing system in ...

Claims

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Application Information

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IPC IPC(8): C12N15/66C12N15/10C12N15/75C12N9/22
CPCC12N15/66C12N15/102C12N15/75C12N9/22C12N2800/80
Inventor 王智文刘丁玉黄灿辛国省陈涛
Owner TIANJIN UNIV
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