A Versatile in Vivo DNA Assembly Toolbox for Fungal Strain Engineering
Zofia Dorota Jarczynska, Katherina García Vanegas, Marcus Deichmann, Christina N. Jensen, Marouschka J. Scheeper, Malgorzata E. Futyma, Tomas Strucko, Fabiano Jares Contesini, Tue Sparholt Jørgensen, Jakob Blæsbjerg Hoof, Uffe Hasbro Mortensen
Abstract
, 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.