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ACS Synth Biol. 2018 Apr 30. doi: 10.1021/acssynbio.7b00456. [Epub ahead of print]

5S rRNA Promoter for Guide RNA Expression Enabled Highly Efficient CRISPR/Cas9 Genome Editing in Aspergillus niger.

Author information

1
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences , Tianjin , 300308 , China.
2
Key Laboratory of Systems Microbial Biotechnology , Chinese Academy of Sciences , Tianjin , 300308 , China.
3
University of Chinese Academy of Sciences , Beijing , 100049 , China.
4
Department Applied and Molecular Microbiology , Institute of Biotechnology, Technische Universit├Ąt Berlin , Berlin , 13355 , Germany.

Abstract

The CRISPR/Cas9 system is a revolutionary genome editing tool. However, in eukaryotes, search and optimization of a suitable promoter for guide RNA expression is a significant technical challenge. Here we used the industrially important fungus, Aspergillus niger, to demonstrate that the 5S rRNA gene, which is both highly conserved and efficiently expressed in eukaryotes, can be used as a guide RNA promoter. The gene editing system was established with 100% rates of precision gene modifications among dozens of transformants using short (40-bp) homologous donor DNA. This system was also applicable for generation of designer chromosomes, as evidenced by deletion of a 48 kb gene cluster required for biosynthesis of the mycotoxin fumonisin B1. Moreover, this system also facilitated simultaneous mutagenesis of multiple genes in A. niger. We anticipate that the use of the 5S rRNA gene as guide RNA promoter can broadly be applied for engineering highly efficient eukaryotic CRISPR/Cas9 toolkits. Additionally, the system reported here will enable development of designer chromosomes in model and industrially important fungi.

KEYWORDS:

5S rRNA; Aspergillus niger; CRISPR/Cas9 system; genome editing

PMID:
29687998
DOI:
10.1021/acssynbio.7b00456

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