Efficient genome editing using tRNA promoter-driven CRISPR/Cas9 gRNA in Aspergillus niger

PloS One
Letian SongAdrian Tsang

Abstract

As a powerful tool for fast and precise genome editing, the CRISPR/Cas9 system has been applied in filamentous fungi to improve the efficiency of genome alteration. However, the method of delivering guide RNA (gRNA) remains a bottleneck in performing CRISPR mutagenesis in Aspergillus species. Here we report a gRNA transcription driven by endogenous tRNA promoters which include a tRNA gene plus 100 base pairs of upstream sequence. Co-transformation of a cas9-expressing plasmid with a linear DNA coding for gRNA demonstrated that 36 of the 37 tRNA promoters tested were able to generate the intended mutation in A. niger. When gRNA and cas9 were expressed in a single extra-chromosomal plasmid, the efficiency of gene mutation was as high as 97%. Co-transformation with DNA template for homologous recombination, the CRISPR/Cas9 system resulted ~42% efficiency of gene replacement in a strain with a functioning non-homologous end joining machinery (kusA+), and an efficiency of >90% gene replacement in a kusA- background. Our results demonstrate that tRNA promoter-mediated gRNA expressions are reliable and efficient in genome editing in A. niger.

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Citations

May 16, 2019·FEMS Microbiology Letters·Belén Adiego-PérezJohn van der Oost
Nov 6, 2018·Folia Microbiologica·Bhagwan Rekadwad, Juan M Gonzalez
Apr 25, 2020·Biotechnology Progress·Zia FatmaHuimin Zhao
Sep 29, 2019·Fungal biology and biotechnology·Tim M van LeeuweArthur F J Ram
Jun 23, 2019·Applied Microbiology and Biotechnology·Yan-Zhen MeiChuan-Chao Dai
Mar 22, 2019·MSphere·Peter B OtoupalJeffrey M Skerker
Feb 13, 2021·BMC Biotechnology·Dede AbdulrachmanKusol Pootanakit
Mar 6, 2021·Microbial Biotechnology·Tania ChroumpiRonald P de Vries
Jun 3, 2021·Journal of Fungi·Gregory EvdokiasIsabelle Benoit-Gelber
Jun 14, 2021·Fungal Genetics and Biology : FG & B·Chenggang Wang, Jeffrey A Rollins
Sep 25, 2021·ACS Synthetic Biology·Cen LiSong Liu
Nov 3, 2021·ACS Synthetic Biology·László MózsikArnold J M Driessen

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Methods Mentioned

BETA
genetic modifications
PCR

Software Mentioned

ARAGORN
SE
Geneious
SPLITSX
tRNAfinder
tRNAscan

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