Simultaneous editing of multiple loci based on the endogenous CRISPR-Cas system of Zymomonas mobilis and its application

Abstract

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. The method mainly includes the following steps: constructing a plasmid containing an artificial CRISPR expression unit; selecting several target editing target sites, and designing gRNA sequences; concatenating several gRNA sequences and constructing them on the plasmid containing an artificial CRISPR expression unit; The DNA sequence is constructed on the vector to obtain the editing plasmid; the editing plasmid is transformed into competent cells for editing. The present invention uses the endogenous CRISPR-Cas system of Zymomonas mobilis to construct a gene editing platform, breaks the limitation of low efficiency of exogenous CRISPR-Cas9 genome editing in this type of strain, and realizes simultaneous rapid and efficient knockout of multiple genes in this strain In addition, promote the development of metabolic engineering, systems 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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