CRISPR/Cas9-mediated genome editing in Penicillium oxalicum and Trichoderma reesei using 5S rRNA promoter-driven guide R
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ORIGINAL RESEARCH PAPER
CRISPR/Cas9-mediated genome editing in Penicillium oxalicum and Trichoderma reesei using 5S rRNA promoterdriven guide RNAs Qi Wang . Qinqin Zhao . Qin Liu . Xin He . Yaohua Zhong . Yuqi Qin . Liwei Gao . Guodong Liu . Yinbo Qu
Received: 19 June 2020 / Accepted: 6 October 2020 Ó Springer Nature B.V. 2020
Abstract Objective To construct convenient CRISPR/Cas9mediated 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 bglucosidase 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%.
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10529-020-03024-7) contains supplementary material, which is available to authorized users. Q. Wang Q. Zhao Q. Liu X. He Y. Zhong Y. Qin L. Gao (&) G. Liu (&) Y. Qu State Key Laboratory of Microbial Technology, National Glycoengineering Research Center, Shandong University, 27 Binhai Road, Qingdao 266237, China e-mail: [email protected] G. Liu e-mail: [email protected]
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. Keywords Genome editing Filamentous fungi Penicillium oxalicum Trichoderma reesei 5S rRNA promoter
Introduction Filamentous fungi are widely used in industry for the production of enzymes, organic acids, and antibiotics. In the recent five years, the CRISPR-based genome editing tools have been developed for many filamentous fungal species (Deng et al. 2017; Krappmann 2017; Shi et al. 2017). With generally higher efficiencies than traditional genetic manipulation methods, the CRISPR-based editing methods have been successfully used for the engineering of industry-relevant fungal strains (Fonseca et al. 2020; Liu et al. 2019; Shi et al. 2019). Ideally, the genome editing methods should be highly efficient, easy to operate, and applicable for multi-gene editing. While the basic principle is similar, the reported genome editing methods in filamentous fungi differ in respect of the forms of CRISPR nuclease delivery, guide RNA expression,
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and donor DNA design. The CRISPR nuclease (mainly Cas9) and guide RNA could be expressed in vivo or preassembled into ribonucleoproteins in vitro (Kwon et al. 2019). For transformant screening, a selectable marker gene is needed, which could be eit
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