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A simple approach to mediate genome editing in the filamentous fungus Trichoderma reesei by CRISPR/Cas9-coupled in vivo gRNA transcription

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Abstract

Objective

To simplify CRISPR/Cas9 genome editing in the industrial filamentous fungus Trichoderma reesei based on in vivo guide RNA (gRNA) transcription.

Results

Two putative RNA polymerase III U6 snRNA genes were identified in the genome of T. reesei QM6a by BLASTN using Myceliophthora. thermophila U6 snRNA gene as the template. The regions approximately 500 bp upstream of two U6 genes were efficient promoters for the in vivo expression of gRNA. The CRISPR system consisting of Cas9 and in vivo synthesized gRNA under control of the T. reesei U6 snRNA promoters was sufficient to cause a frameshift mutation in the ura5 gene via non-homologous end-joining-mediated events.

Conclusions

We report a simple gene editing method using a CRISPR/Cas9-coupled in vivo gRNA transcription system in T. reesei.

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Acknowledgements

The project funded by China Postdoctoral Science Foundation funded project (No. 2019M661402), the Open Funding Project of the State Key Laboratory of Bioreactor Engineering, and the Fundamental Research Funds for the Central Universities (No. 222201714053). We thank Professor Zihua Zhou for donating the T. reesei 6a-pc strain.

Supporting information

Supplementary Table 1—Primers and oligonucleotides used in this study.

Supplementary Table 2—The mutation efficiency in this study.

Sequence data—  > gRNA-ura5. > PU6-1. > PU6-2.

Supplementary Fig. 1—The procedure of T. reesei transformation.

Supplementary Fig. 2—Transcription of the gRNA in Dura1 and Dura2. The relative transcriptional levels of TrU6-1, TrU6-2, and gRNA in T. reesei mutants in glucose were analyzed by RT-qPCR. The expression of each tested gene was normalized to that of the internal control. The housekeeping gene sar1 was used as the internal control for normalization. The gene expression ratio in Dura1 was set to 1. Three independent experiments with three biological replicates each were performed. Values are the means ± SD of the results of three independent experiments.

Author information

Authors and Affiliations

  1. State Key Lab of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, 130 Meilong Road, P.O.B. 311, Shanghai, 200237, China

    Chuan Wu, Yumeng Chen, Yifei Qiu, Xiao Niu, Jiehui Chen, Hong Yao, Wei Wang & Yushu Ma

  2. The First Affiliated Middle School of East China Normal University, Shanghai, 200086, China

    Ningjian Zhu

Authors
  1. Chuan Wu
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  2. Yumeng Chen
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  3. Yifei Qiu
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  4. Xiao Niu
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  5. Ningjian Zhu
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  6. Jiehui Chen
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  7. Hong Yao
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  8. Wei Wang
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  9. Yushu Ma
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Contributions

WW initiated, designed and coordinated the study and reviewed the manuscript. CW designed and carried out experiments and measurements. YC interpreted experimental data. YQ, JC, XN, HY, NZ cultured and counted fungi strains. YM supported the research funding. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Wei Wang or Yushu Ma.

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Wu, C., Chen, Y., Qiu, Y. et al. A simple approach to mediate genome editing in the filamentous fungus Trichoderma reesei by CRISPR/Cas9-coupled in vivo gRNA transcription. Biotechnol Lett 42, 1203–1210 (2020). https://doi.org/10.1007/s10529-020-02887-0

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  • DOI: https://doi.org/10.1007/s10529-020-02887-0

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