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

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...

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Bibliographic Details
Published in:Biotechnology letters 2020-07, Vol.42 (7), p.1203-1210
Main Authors: Wu, Chuan, Chen, Yumeng, Qiu, Yifei, Niu, Xiao, Zhu, Ningjian, Chen, Jiehui, Yao, Hong, Wang, Wei, Ma, Yushu
Format: Article
Language:English
Subjects:
Applied Microbiology
Biochemistry
Biomedical and Life Sciences
Biotechnology
CRISPR
CRISPR-Cas Systems - genetics
DNA-directed RNA polymerase
Frameshift mutation
Fungi
Gene Editing - methods
Genes
Genetic modification
Genome editing
Genome, Fungal - genetics
Genomes
gRNA
Homology
Hypocreales - genetics
In vivo methods and tests
Life Sciences
Microbiology
Mutation
Non-homologous end joining
Original Research Paper
Promoter Regions, Genetic - genetics
Promoters
Ribonucleic acid
RNA
RNA polymerase
RNA, Guide, CRISPR-Cas Systems - genetics
snRNA
Transcription
Transcription, Genetic - genetics
Trichoderma reesei
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Summary: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 .
ISSN:0141-5492
1573-6776
DOI:10.1007/s10529-020-02887-0