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Summary  The filamentous fungus  Ashbya gossypii  is currently used for the industrial production of vitamin B2. Furthermore, the ability of  A. gossypii  to grow using low‐cost substrates together with the inexpensive downstream processing makes this fungus an attractive biotechnological chassis. Indeed, the production in  A. gossypii  of other high‐added value compounds such as folic acid, nucleosides and biolipids has been described. Hence, the development of new methods to expand the molecular toolkit for  A. gossypii  genomic manipulation constitutes an important issue for the biotechnology of this fungus. In this work, we present a one‐vector CRISPR/Cas9 system for genomic engineering of  A. gossypii . We demonstrate the efficiency of the system as a marker‐less approach for nucleotide deletions and substitutions both with visible and invisible phenotypes. Particularly, the system has been validated for three types of genomic editions: gene inactivation, the genomic erasure of  lox P scars and the introduction of point mutations. We anticipate that the use of the CRISPR/Cas9 system for  A. gossypii  will largely contribute to facilitate the genomic manipulations of this industrial fungus in a marker‐less manner.

One‐vector CRISPR/Cas9 genome engineering of the industrial fungus Ashbya gossypii