A Versatile in Vivo DNA Assembly Toolbox for Fungal Strain Engineering

Zofia Dorota Jarczynska, Katherina Garcia Vanegas, Marcus Deichmann, Christina Nørskov Jensen, Marouschka Jasmijn Scheeper, Malgorzata Ewa Futyma, Tomas Strucko, Fabiano Jares Contesini, Tue Sparholt Jørgensen, Jakob Blæsbjerg Hoof, Uffe Hasbro Mortensen*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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Efficient homologous recombination in baker's yeast allows accurate fusion of DNA fragments via short identical sequence tags in vivo. Eliminating the need for an Escherichia coli cloning step speeds up genetic engineering of this yeast and sets the stage for large high-throughput projects depending on DNA construction. With the aim of developing similar tools for filamentous fungi, we first set out to determine the genetic- and sequence-length requirements needed for efficient fusion reactions, and demonstrated that in nonhomologous end-joining deficient strains of Aspergillus nidulans, efficient fusions can be achieved by 25 bp sequence overlaps. Based on these results, we developed a novel fungal in vivo DNA assembly toolbox for simple and flexible genetic engineering of filamentous fungi. Specifically, we have used this method for construction of AMA1-based vectors, complex gene-targeting substrates for gene deletion and gene insertion, and for marker-free CRISPR based gene editing. All reactions were done via single-step transformations involving fusions of up to six different DNA fragments. Moreover, we show that it can be applied in four different species of Aspergilli. We therefore envision that in vivo DNA assembly can be advantageously used for many more purposes and will develop into a popular tool for fungal genetic engineering.
Original languageEnglish
JournalACS Synthetic Biology
Issue number10
Pages (from-to)3251-3263
Number of pages13
Publication statusPublished - 2022


  • Filamentous fungi
  • In vivo
  • DNA assembly
  • Gene targeting
  • Gene expression


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