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Background  The CRISPR/Cas9 is currently the predominant technology to enhance the genome editing efficiency in eukaryotes. Established tools for many fungal species exist while most of them are based on in vivo expressed Cas9 and guide RNA (gRNA). Alternatively, in vitro assembled Cas9 and gRNA ribonucleoprotein complexes can be used in genome editing, however, only a few examples have been reported in fungi. In general, high-throughput compatible transformation workflows for filamentous fungi are immature.    Results  In this study, a CRISPR/Cas9 facilitated transformation and genome editing method based on in vitro assembled ribonucleoprotein complexes was developed for the filamentous fungus  Aspergillus niger . The method was downscaled to be compatible with 96-well microtiter plates. The optimized method resulted in 100% targeting efficiency for a single genomic target. After the optimization, the method was demonstrated to be suitable for multiplexed genome editing with two or three genomic targets in a metabolic engineering application. As a result, an  A. niger  strain with improved capacity to produce galactarate, a potential chemical building block, was generated.    Conclusions  The developed microtiter plate compatible CRISPR/Cas9 method provides a basis for high-throughput genome editing workflows in  A. niger  and other related species. In addition, it improves the cost-effectiveness of CRISPR/Cas9 genome editing methods in fungi based on in vitro assembled ribonucleoproteins. The demonstrated metabolic engineering example with multiplexed genome editing highlights the applicability of the method.    Electronic supplementary material  The online version of this article (10.1186/s40694-019-0066-9) contains supplementary material, which is available to authorized users.

Development of microtiter plate scale CRISPR/Cas9 transformation method for Aspergillus niger based on in vitro assembled ribonucleoprotein complexes