Efficient Genome Editing Using TRNA Promoter-driven CRISPR/Cas9 GRNA in Aspergillus Niger

Overview

Journal PLoS One

Specialties General Medicine
Science

Date 2018 Aug 25

PMID 30142205

Citations 54

Authors

Letian Song

Jean-Paul Ouedraogo

Magdalena Kolbusz

Thi Truc Minh Nguyen

Adrian Tsang

Affiliations

Soon will be listed here.

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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