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Method for highly efficiently deleting large fragments of genome based on endogenous CRISPR-Cas system of zymomonas mobilis and application thereof

A technology of Zymomonas and deletion method, applied in the field of efficient deletion of large genome fragments, can solve the problems of host cell toxicity and difficult application, and achieve the effect of avoiding cytotoxicity and efficient deletion of large genome fragments

Active Publication Date: 2019-11-05
武汉睿嘉康生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, with the deepening of research, more and more results show that heterologous expression of these nucleases can cause varying degrees of cytotoxicity to the host
This may be one of the main reasons why relevant applications have been difficult to carry out in Zymomonas mobilis so far

Method used

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  • Method for highly efficiently deleting large fragments of genome based on endogenous CRISPR-Cas system of zymomonas mobilis and application thereof
  • Method for highly efficiently deleting large fragments of genome based on endogenous CRISPR-Cas system of zymomonas mobilis and application thereof
  • Method for highly efficiently deleting large fragments of genome based on endogenous CRISPR-Cas system of zymomonas mobilis and application thereof

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Embodiment 1

[0031] Example 1 Construction of Zymomonas mobilis Endogenous CRISPR-Cas Genome Editing System

[0032] (1) CRISPR-Cas phylogenetic analysis of the genome encoding of Zymomonas mobilis

[0033] 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.

[0034] Taking Zymomonas mobilis Z.mobilis 4 as the model strain, the sequencing data of Z.mobilis 4 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 sequenc...

Embodiment 2

[0065] Example 2 Application of the method for efficient deletion of large genome fragments based on the endogenous CRISPR-Cas system of Zymomonas mobilis

[0066] In the present invention, bioinformatics methods are firstly used to determine the essential genes that need to be retained and the non-essential genes that can be deleted, and select a non-essential gene with a length of 10 kb as the target knockout large fragment. Then design guide RNA and donor DNA sequences. Finally, the artificial CRISPR cluster expression module and donor DNA sequence were loaded on the plasmid, and the plasmid was electrotransformed into Zymomonas mobilis cells to complete the editing. Schematic diagram see Figure 5 As shown, the specific experimental scheme is as follows:

[0067] (1) Selection of target knockout large fragments

[0068] Through bioinformatics analysis, a non-essential gene ZMO1815-ZMO1822 (10,021bp) was found on the genome of the bacteria, which can be used as the targe...

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Abstract

The invention belongs to the technical field of genetic engineering, and in particular, relates to a method for highly efficiently deleting large fragments of a genome based on an endogenous CRISPR-Cas system of zymomonas mobilis and an application thereof. The method includes the steps: constructing a plasmid containing an artificial CRISPR expression unit; determining a large-fragment editing target site, selecting a guideRNA sequence for the editing target site and designing a primer; constructing the guideRNA primer sequence to the plasmid containing the artificial CRISPR expression unit,and constructing a vector; constructing a donor DNA sequence to the vector, to obtain an editing plasmid; and transforming the editing plasmid into a receptive cell, and editing. With a high-throughput gene editing platform based on the endogenous CRISPR-Cas system of zymomonas mobilis, the editing purpose of deleting the large fragments of the genome of the strain is achieved, and the developmentof metabolic engineering, system biology and synthetic biology is promoted.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for efficiently deleting large genome fragments 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] Because Zymomonas mobilis can produce alcohol naturally, it ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/90C12N9/22C12R1/01
CPCC12N9/22C12N15/74C12N15/902
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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