Application of high-throughput screening tool for enabling Escherichia coli to obtain effective NHEJ system in Escherichia coli gene editing

Abstract

The invention provides application of a high-throughput screening tool for enabling escherichia coli to obtain an effective NHEJ system in escherichia coli gene editing. The high-throughput screening tool for enabling the Escherichia coli to obtain the effective NHEJ system comprises a pDual-Cas9-Parental plasmid vector and a high-throughput screening vector, wherein the pDual-Cas9-Parental plasmid vector contains a DNA helicase gene, a replicon, an antibiotic resistance gene, a nuclease gene, an araC gene, an arabinose promoter and an IIs type restriction enzyme recognition site; and the pDual-sgRNA-lacZ plasmid vector contains an sgRNA sequence of a targeted lacZ gene, a strong promoter for constitutive expression, a replicon and an antibiotic resistance gene. The NHEJ system obtained through screening has good connection efficiency in escherichia coli, efficient gene editing can be carried out, and the application prospect is wide.

Description

technical field [0001] The invention belongs to the technical field of gene editing, and in particular relates to the application of a high-throughput screening tool for obtaining an effective NHEJ system for Escherichia coli in gene editing of Escherichia coli. Background technique [0002] CRISPR-Cas9 is an adaptive immune defense system formed during the long-term evolution of bacteria and archaea, which can be used to fight against invading viruses and foreign DNA. The system can recognize the target sequence and cut the target sequence to form DSB (double-stranded breaks, double-strand break). After the genome of an organism breaks and forms a DSB, DNA repair will be initiated through the NHEJ (non-homologous end joining) pathway or through the HR (homologous recombination, homologous recombination) pathway in the presence of a homologous template . CRISPR-Cas9 technology has been widely used in genome editing of eukaryotic and prokaryotic organisms, including human, ...

AI Technical Summary

Problems solved by technology

Although both methods are capable of genome editing, mutation rates are generally low

(3) Use the CRISPR-Cas9 system to simultaneously express the three proteins of the Red system, and perform homologous recombination gene editing in the presence of homologous templates. This method has the advantage of high gene editing efficiency, but this method is applied on a genome scale gene editing is limited by the extremely complex construction of template plasmids

Although Escherichia coli can obtain the ability of non-homologous end joining by expressing the exogenous NHEJ system, the inherent homologous recombination of Escherichia coli itself is in a competitive relationship with the introduced NHEJ system. If the NHEJ system connection efficiency is low, homologous recombination Repair would then predominate, causing the connection to the NHEJ system to "fail"

[0007] Although Tianyuan Su et al., Xuan Zheng et al. (X.Zheng,S.Li,G.Zhao,J.Wang.(2017).A efficient system for deletion of large DNA fragments in Escherichia coli via introduction of both Cas9 and the non-homologous end joining system from Mycobacterium smegmatis, BBRC.(2017) 1-7) successfully introduced the NHEJ system into E. coli by making E. coli express Ku and ligD proteins from other microorganisms, but so far there is still no efficient way to make E. coli obtain the NHEJ system and Methods for gene editing

Images