Efficient assembly of long DNA fragments and multiple genes with improved nickase‐based cloning and Cre/loxP recombination. Issue 10 (16th July 2022)

• Record Type: Journal Article
• Authors: Zhao, Yanchang; Han, Jingluan; Tan, Jiantao; Yang, Yaqian; Li, Shuangchun; Gou, Yajun; Luo, Yuyu; Li, Tie; Xiao, Wenyu; Xue, Yang; Hao, Yu; Xie, Xianrong; Liu, Yao‐Guang; Zhu, Qinlong
• Is Part Of: Plant biotechnology journal. Volume 20:Issue 10(2022)
• Page Start: 1983
• Page End: 1995
• Publication Date: 2022-07-16
• Subjects: DNA assembly -- multigene stacking -- nicking endonucleases -- synthetic biology; Plant biotechnology -- Periodicals; Plant genetic engineering -- Periodicals; 630.272
• DOI: 10.1111/pbi.13882
• Languages: English
• ISSNs: 1467-7644

Abstract

Summary: Functional genomics, synthetic biology and metabolic engineering require efficient tools to deliver long DNA fragments or multiple gene constructs. Although numerous DNA assembly methods exist, most are complicated, time‐consuming and expensive. Here, we developed a simple and flexible strategy, unique nucleotide sequence‐guided nicking endonuclease (UNiE)‐mediated DNA assembly (UNiEDA), for efficient cloning of long DNAs and multigene stacking. In this system, a set of unique 15‐nt 3′ single‐strand overhangs were designed and produced by nicking endonucleases (nickases) in vectors and insert sequences. We introduced UNiEDA into our modified Cre/ loxP recombination‐mediated TransGene Stacking II (TGSII) system to generate an improved multigene stacking system we call TGSII‐UNiE. Using TGSII‐UNiE, we achieved efficient cloning of long DNA fragments of different sizes and assembly of multiple gene cassettes. Finally, we engineered and validated the biosynthesis of betanin in wild tobacco ( Nicotiana benthamiana ) leaves and transgenic rice ( Oryza sativa ) using multigene stacking constructs based on TGSII‐UNiE. In conclusion, UNiEDA is an efficient, convenient and low‐cost method for DNA cloning and multigene stacking, and the TGSII‐UNiE system has important application prospects for plant functional genomics, genetic engineering and synthetic biology research.