基因编辑技术在大肠杆菌中的应用

Application of gene editing technology in Escherichia coli

摘要:

利用基因编辑技术对大肠杆菌基因组进行改造可以研究基因功能,或改变其代谢途径大量生产原本成本较高的产物,从而获得可以生产特定产物的遗传稳定性工程菌株。目前可以对细菌基因组编辑的方法有Red同源重组、CRISPR/Cas9技术等。Red同源重组是比较传统的基因编辑技术,应用广泛,但编辑效率受整合片段大小的限制,基因编辑过程比较烦琐,且重组后基因组会有FRT位点残留。CRISPR/Cas9技术应用广泛,可靶向基因组特定位置进行编辑,但需要根据编辑位点设计特定的DNA打靶片段。随着人们对这两种技术越来越深入的研究,衍生出了多种复合基因编辑技术。如Red同源重组和归巢核酸内切酶Ⅰ-SceⅠ的联合运用,Red同源重组和CRISPR/Cas9的联合运用等。本文总结了常用的几种基因编辑技术及复合基因编辑技术的基本原理及在大肠杆菌中的应用,可为原核生物基因编辑方法的选择提供依据。

关键词:基因编辑;工程菌株;代谢途径

Abstract:

Gene editing technology can be used to modify the genome of Escherichia coli for the investigation of gene functions,or to change the metabolic pathways for the efficient production of high-value products in engineered strains with genetic stability.A variety of gene editing technologies have been applied in prokaryotes,such as λ-Red homologous recombination and CRISPR/Cas9.As a traditional gene editing technique,λ-Red recombination is widely used.However,it has a few shortcomings,such as the limited integration efficiency by the integrated fragment size,the cumbersome gene editing process,and the FRT scar in the genome after recombination.CRISPR/Cas9 is widely used for genome editing at specific sites,which requires specific DNA segments according to the editing site.As the understanding of the two technologies deepens,a variety of composite gene editing techniques have been developed,such as the application of λ-Red homologous recombination in combination with homing endonucleaseⅠ-SceⅠ or CRISPR/Cas9.In this review,we summarized the basic principles of common gene editing techniques and composite gene editing techniques,as well as their applications in Escherichia coli,which can provide a basis for the selection of gene editing methods in prokaryotes.

Key words:gene editing;engineered strain;metabolic pathways