Unknown

Dataset Information

0

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


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.

SUBMITTER: Song L 

PROVIDER: S-EPMC6108506 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

altmetric image

Publications

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

Song Letian L   Ouedraogo Jean-Paul JP   Kolbusz Magdalena M   Nguyen Thi Truc Minh TTM   Tsang Adrian A  

PloS one 20180824 8


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 c  ...[more]

Similar Datasets

| S-EPMC6400946 | biostudies-literature
| S-EPMC6829922 | biostudies-literature
| S-EPMC5159642 | biostudies-literature
| S-EPMC6336261 | biostudies-literature
| S-EPMC5806293 | biostudies-literature
| S-EPMC4544769 | biostudies-literature
| S-EPMC9241840 | biostudies-literature
| S-EPMC5852178 | biostudies-literature
| S-EPMC6429391 | biostudies-literature
| S-EPMC6180501 | biostudies-literature