Open AccessEfficient assembly of long DNA fragments and multiple genes with improved nickase‐based cloning and Cre/ loxP recombination
Author(s) -
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 YaoGuang,
Zhu Qinlong
Publication year - 2022
Publication title -
plant biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.525
H-Index - 115
eISSN - 1467-7652
pISSN - 1467-7644
DOI - 10.1111/pbi.13882
Subject(s) - biology , cloning (programming) , gene , dna , genetics , cre lox recombination , microbiology and biotechnology , recombination , transgene , genetically modified mouse , computer science , programming language
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.