Abstract
BackgroundThe 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.ResultsIn 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.ConclusionsThe 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.
Highlights
The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 is currently the predominant technology to enhance the genome editing efficiency in eukaryotes
Down‐scaled transformation reaction for A. niger We aimed to develop a fast and robust CRISPR/Cas9 genome editing method for A. niger which is based on in vitro assembled RNP complexes
The downscaled reaction volume would require less protoplasts, RNP complex and donor DNA to be used in the transformation which will decrease the cost per transformation in our case by approximately 90%
Summary
The CRISPR/Cas 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 Cas and guide RNA (gRNA). The use of programmable nucleases, especially the CRISPR/Cas technology [1, 2] derived from a prokaryotic immune system, has greatly improved the efficiency of targeted genome editing in eukaryotes [3, 4]. The CRISPR/Cas is currently the predominant programmable nuclease system to generate intentional DSBs in genome editing [3, 4]. The system consists of Cas nuclease, typically derived from Streptococcus pyogenes, and the guide RNA (gRNA) components CRISPR-RNA (crRNA) and trans-activating crRNA (tracrRNA) [1, 2]. The DNA endonuclease activity of Cas generates a DSB about 3–4 nucleotides upstream of the PAM sequence [1, 2]
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