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

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...

AI Technical Summary

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 for prokaryotic cells

[0005] Cas9 has obvious cytotoxicity to Zymomonas mobilis, and expressing Cas9 on a plasmid will cause a thousand-fold reduction in transformation efficiency. For data comparison, see figure 1

In addition, in order to achieve simultaneous editing of multiple sites, multiple gRNA expression cassettes need to be cloned and expressed in clusters, which is complicated and time-consuming

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