ArticlePlant biotechnology journal2022
Efficient assembly of long DNA fragments and multiple genes with improved nickase-based cloning and Cre/loxP recombination.
Article in Plant biotechnology journal, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 12 papers.
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12 citing papers in PubMed, 24 citations in OpenAlex.
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- Transgene-Killer-CRISPR version 2 (TKC2) eliminates occasional transgene escape by coupling with a RUBY reporter.Plant biotechnology journal · 2025Article
- DASH: a versatile and high-capacity gene stacking system for plant synthetic biology.Plant biotechnology journal · 2025Review
- A RUBY Reporter for Efficient Banana Transformation and Development of Betalain-RichInternational journal of molecular sciences · 2025Article
- Enhancement of Dendrobine Production by CRISPR/Act3.0-Mediated Transcriptional Activation of Multiple Endogenous Genes inInternational journal of molecular sciences · 2025Article
- Multiplex Expression Cassette Assembly: A flexible and versatile method for building complex genetic circuits in conventional vectors.Plant biotechnology journal · 2024Article
- Metabolic Pathway Engineering Improves Dendrobine Production inInternational journal of molecular sciences · 2023Article
- From qualitative to quantitative: the state of the art and challenges for plant synthetic biology.Quantitative biology (Beijing, China) · 2023Review
- Generating colorful carrot germplasm through metabolic engineering of betalains pigments.Horticulture research · 2023Article
- Anthocyanins in Plant Food: Current Status, Genetic Modification, and Future Perspectives.Molecules (Basel, Switzerland) · 2023Review
- Pyramiding stacking of multigenes (PSM): a simple, flexible and efficient multigene stacking system based on Gibson assembly and gateway cloning.Frontiers in bioengineering and biotechnology · 2023Article
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Authors and funding
14 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
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.
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