Development of a plasmid free CRISPR-Cas9 system for the genetic modification of Mucor circinelloides

Gábor Nagy,Amanda Grace Vaz,Tamás Papp,Csilla Szebenyi,Árpád Csernetics,Eszter Judit Tóth,Csaba Vágvölgyi

doi:10.1038/s41598-017-17118-2

Gábor Nagy, Amanda Grace Vaz + Show 5 more

Open Access

https://doi.org/10.1038/s41598-017-17118-2

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Abstract

Mucor circinelloides and other members of Mucorales are filamentous fungi, widely used as model organisms in basic and applied studies. Although genetic manipulation methods have been described for some Mucoral fungi, construction of stable integrative transformants by homologous recombination has remained a great challenge in these organisms. In the present study, a plasmid free CRISPR-Cas9 system was firstly developed for the genetic modification of a Mucoral fungus. The described method offers a rapid but robust tool to obtain mitotically stable mutants of M. circinelloides via targeted integration of the desired DNA. It does not require plasmid construction and its expression in the recipient organism. Instead, it involves the direct introduction of the guide RNA and the Cas9 enzyme and, in case of homology directed repair (HDR), the template DNA into the recipient strain. Efficiency of the method for non-homologous end joining (NHEJ) and HDR was tested by disrupting two different genes, i.e. carB encoding phytoene dehydrogenase and hmgR2 encoding 3-hydroxy-3-methylglutaryl-CoA reductase, of M. circinelloides. Both NHEJ and HDR resulted in stable gene disruption mutants. While NHEJ caused extensive deletions upstream from the protospacer adjacent motif, HDR assured the integration of the deletion cassette at the targeted site.

Highlights

Members of the order Mucorales constitute a remarkable group of filamentous fungi
M. circinelloides f. lusitanicus used in the present study has been involved in several studies addressed to the role of RNA interference in the fungal cell[15], mechanism and role of morphological dimorphism[7,16], production of enzymes, carotenoids and other metabolites[1] and the genetic and molecular background of the pathogenicity of Mucoral fungi[7,17,18]
Plasmid DNAs introduced into Mucoral fungi generally do not integrate into the genome even if they harbor long sequences homologous with the targeted sites and remain autonomously replicating elements frequently causing mitotic instability of the transformants[21]. Such plasmids often form high molecular weight concatenated structures, which make difficult the interpretation of the results of the transformation experiments[19,20,21]. The latter phenomenon is explained by the activity of the non-homologous end joining (NHEJ), which is the dominant mechanism of double-strand break repair over homologous recombination in these fungi[21]

Summary

Introduction

Members of the order Mucorales constitute a remarkable group of filamentous fungi. Some of them have biotechnological importance as producers of extracellular hydrolytic enzymes (primarily of lipases and proteases), organic acids (e.g. lactic, fumaric and malic acids), alcohol and carotenoids[1,2], others are used in starter cultures of oriental food fermentations[3]. Plasmid DNAs introduced into Mucoral fungi generally do not integrate into the genome even if they harbor long sequences homologous with the targeted sites and remain autonomously replicating elements frequently causing mitotic instability of the transformants[21]. Such plasmids often form high molecular weight concatenated structures, which make difficult the interpretation of the results of the transformation experiments[19,20,21]. With this method the off-target effect is avoidable but the RNP can be degraded [43]

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Results

Conclusion