Abstract
Objective
To construct convenient CRISPR/Cas9-mediated genome editing systems in industrial enzyme-producing fungi Penicillium oxalicum and Trichoderma reesei.
Results
Employing the 5S rRNA promoter from Aspergillus niger for guide RNA expression, the β-glucosidase gene bgl2 in P. oxalicum was deleted using a donor DNA carrying 40-bp homology arms or a donor containing no selectable marker gene. Using a markerless donor DNA as editing template, precise replacement of a small region was achieved in the creA gene. In T. reesei, the A. niger 5S rRNA promoter was less efficient than that in P. oxalicum when used for gene editing. Using a native 5S rRNA promoter, stop codons were introduced into the lae1 coding region using a markerless donor DNA with an editing efficiency of 36.67%.
Conclusions
Efficient genome editing systems were developed in filamentous fungi P. oxalicum and T. reesei by using heterologous or native 5S rRNA promoters for guide RNA expression.
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Acknowledgements
We thank Xiaomei Zheng, Jibin Sun and Weifeng Liu for providing plasmids. This work was supported by National Key R&D Program of China Grant (2018YFA0900500), Major Basic Research Program of Shandong Provincial Natural Science Foundation (ZR2019ZD19), the Key Research and Development Project of Shandong Province (2019JZZY020223 and 2019JZZY020807), and the Young Scholars Program of Shandong University (YSPSDU) (to G. L.).
Supplementary Materials
Fig. S1 Plasmids containing Cas9 expression cassettes.
Fig. S2 The growth of P. oxalicum M12 and 9-10 on 2% (w/v) glucose agar plate.
Fig. S3 Diagnostic PCR analysis of P. oxalicum bgl2 deletion transformants.
Fig. S4 Diagnostic PCR analysis of P. oxalicum creA editing transformants.
Table S1 Sequences of protospacers in this study.
Table S2 Primers used in this study.
Table S3 Sequences of Cas9 expression cassettes.
Table S4 Sequences of sgRNA expression cassettes in this study.
Table S5 Sequences of donor DNAs in this study.
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Wang, Q., Zhao, Q., Liu, Q. et al. CRISPR/Cas9-mediated genome editing in Penicillium oxalicum and Trichoderma reesei using 5S rRNA promoter-driven guide RNAs. Biotechnol Lett 43, 495–502 (2021). https://doi.org/10.1007/s10529-020-03024-7
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DOI: https://doi.org/10.1007/s10529-020-03024-7