一种用于归巢内切核酸酶表征与工程改造的双质粒细菌筛选系统。

A two-plasmid bacterial selection system for characterization and engineering of homing endonucleases.

作者信息

Sun Ning, Zhao Huimin

机构信息

Departments of Chemical and Biomolecular Engineering, Chemistry, Biochemistry, and Bioengineering, Institute for Genomic Biology, Center for Biophysic and Computational, Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA.

出版信息

Methods Mol Biol. 2014;1123:87-96. doi: 10.1007/978-1-62703-968-0_7.

DOI:10.1007/978-1-62703-968-0_7

PMID:24510262

Abstract

Homing endonucleases recognize long DNA sequences and generate site-specific DNA double-stranded breaks. They can serve as a powerful genomic modification tool in various industrial and biomedical applications. Here, we describe a two-plasmid bacterial selection system for characterization and engineering of homing endonucleases. This selection system couples the DNA cleavage activity of a homing endonuclease with the survival of host cells. Therefore, it can be used for assaying in vivo activity of homing endonucleases. Moreover, due to its high sensitivity, it can be applied for directed evolution of homing endonucleases with altered sequence specificity.

摘要

归巢内切酶识别长DNA序列并产生位点特异性DNA双链断裂。它们可作为一种强大的基因组修饰工具，应用于各种工业和生物医学领域。在此，我们描述了一种用于归巢内切酶表征和工程改造的双质粒细菌筛选系统。该筛选系统将归巢内切酶的DNA切割活性与宿主细胞的存活联系起来。因此，它可用于检测归巢内切酶的体内活性。此外，由于其高灵敏度，它可应用于对具有改变的序列特异性的归巢内切酶进行定向进化。