Multi-gene locus simultaneous editing method based on endogenous CRISPR-Cas system of zymomonas mobilis and application thereof

A Zymomonas multigene technology, applied in the field of genetic engineering, can solve the problems of host cytotoxicity, complicated operation procedures, reduced transformation efficiency, etc., and achieve the effects of avoiding cytotoxicity, avoiding limited available selection markers, and shortening the cycle.

Active Publication Date: 2019-10-15
武汉睿嘉康生物科技有限公司
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  • Abstract
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  • Application Information

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Problems solved by technology

However, since the currently commonly used Cas9 is a large nucleic acid protein (more than 1000 amino acids) with multiple domains, there is a potential difficulty in transporting it into the target cell. More importantly, with the deepening of research, more and more The results show that heterologous expression of these nucleases can cause varying degrees of cytotoxicity to the host, especially fo

Method used

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  • Multi-gene locus simultaneous editing method based on endogenous CRISPR-Cas system of zymomonas mobilis and application thereof
  • Multi-gene locus simultaneous editing method based on endogenous CRISPR-Cas system of zymomonas mobilis and application thereof
  • Multi-gene locus simultaneous editing method based on endogenous CRISPR-Cas system of zymomonas mobilis and application thereof

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Embodiment 1

[0038] Example 1 Construction of Zymomonas mobilis Endogenous CRISPR-Cas Genome Editing System

[0039] (1) CRISPR-Cas phylogenetic analysis of the genome encoding of Zymomonas mobilis

[0040] The premise of the present invention is that the research strain needs to encode the CRISPR-Cas system itself and have DNA splicing activity, which requires the host genome to encode the CRISPR cluster and the complete Cas protein system.

[0041] Taking Zymomonas mobilis Z.mobilis 4 as the model strain, the sequencing data of Z.mobilis ZM4 were analyzed. The results showed that the genome of the strain encoded four CRISPR structural sequences. According to their sequence in the genome, we sequenced them named CRISPR1-CRISPR4, see figure 1. CRISPR1 occupies the 113,783-114,170 region of the genome and contains 7 repeat sequences; CRISPR2 occupies the 1,244,355-1,245,866 region and contains 9 repeat sequences; CRISPR3 occupies the 1,598,754-1,599,144 region and contains 7 repeat sequen...

Embodiment 2

[0072] Example 2 Application of Zymomonas mobilis-based endogenous CRISPR-Cas system in simultaneous editing of multiple gene loci

[0073] The present invention first selects three different editing sites on the genome to design gRNA, and then designs the corresponding donor DNA. Then, construct a gene editing plasmid, construct an artificial CRISPR cluster on the plasmid for simultaneous expression of the corresponding gRNA, and at the same time, clone the donor DNA into the same editing plasmid. Finally, the editing plasmid was electrotransformed into Zymomonas mobilis cells, and the target strain after accurate editing was screened and identified. The specific operation is as follows.

[0074] (1) Select the knockout target site

[0075] The multi-site gene editing object of the present invention is genome-encoded CRISPR genes 1-4 (CRISPR1-4), wherein CRISPR3 and 4 are located adjacent to each other on the genome, and they are knocked out as an editing target.

[0076] ...

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Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a multi-gene locus simultaneous editing method based on an endogenous CRISPR-Cas system of zymomonas mobilis and application thereof. The method mainly comprises the following steps of: constructing a plasmid containing an artificial CRISPR expression unit; selecting a plurality of target editing target loci, and designing a gRNA sequence; connecting a plurality of gRNA sequences in series, and constructing the gRNA sequences on the plasmid containing the artificial CRISPR expression unit; constructing a donor DNA sequence on a vector to obtain an edited plasmid; and transforming the edited plasmid into competent cells for editing. The method constructs a gene editing platform by utilizing theendogenous CRISPR-Cas system of zymomonas mobilis, breaks the limitation of low efficiency of exogenous CRISPR-Cas9 genome editing in the strain, realizes rapid and efficient knockout of multiple genes in the strain, and promotes the development of metabolic engineering, systemic biology and synthetic biology.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a simultaneous editing method of multiple gene sites based on the endogenous CRISPR-Cas system of Zymomonas mobilis and its application. Background technique [0002] In recent years, the use of microorganisms for metabolic engineering, systems biology, and synthetic biology has made good progress. For the rational design and construction of microbial cell factories, living cells or enzymes are used to treat renewable biomass, such as It provides an important theoretical basis for the transformation of cellulose and other substances, the production of bioenergy, and the industrialization of biosmelting. Bioenergy regeneration is one of the effective means to solve the problems of resource and energy shortage and serious environmental pollution that human beings are currently facing. [0003] Conventional genetic manipulation methods are cumbersome and hav...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N1/21C12R1/01
CPCC12N9/22C12N15/113C12N15/74
Inventor 彭文舫杨世辉郑艳丽易犁马立新
Owner 武汉睿嘉康生物科技有限公司
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