Journal
ACS SYNTHETIC BIOLOGY
Volume 8, Issue 7, Pages 1568-1574Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.7b00456
Keywords
SS rRNA; Aspergillus niger; CRISPR/Cas9 system; genome editing
Categories
Funding
- National Natural Science Foundation of China [31700085, 31370113, 31370829]
- Chinese Academy of Sciences President's International Fellowship Initiative CAS PIFI [2018VBA0013, 2018PB0036]
- Marie Curie International Training Network QuantFung FP7-People-2013-ITN [607332]
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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 SS 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 Bl. 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.
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