Format

Send to

Choose Destination
Fungal Genet Biol. 2016 Jan;86:47-57. doi: 10.1016/j.fgb.2015.12.007. Epub 2015 Dec 14.

Highly efficient CRISPR mutagenesis by microhomology-mediated end joining in Aspergillus fumigatus.

Author information

1
Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Microbiology, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China.
2
Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Microbiology, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China. Electronic address: linglu@njnu.edu.cn.

Abstract

Filamentous fungi have a dominant nonhomologous-end joining (NHEJ) DNA repair pathway, which results in the majority of transformed progenies having random heterologous insertion mutagenesis. Thus, lack of a versatile genome-editing tool prevents us from carrying out precise genome editing to explore the mechanism of pathogenesis. Moreover, clinical isolates that have a wild-type ku80 background without any selection nutrition marker especially suffer from low homologous integration efficiency. In this study, we have established a highly efficient CRISPR mutagenesis system to carry out precise and efficient in-frame integration with or without marker insertion with approximately 95-100% accuracy via very short (approximately 35-bp) homology arms in a process referred to as microhomology-mediated end joining (MMEJ). Based on this system, we have successfully achieved an efficient and precise integration of an exogenous GFP tag at the predicted site without marker insertion and edited a conidial melanin gene pksP and a catalytic subunit of calcineurin gene cnaA at multiple predicted sites with or without selection marker insertion. Moreover, we found that MMEJ-mediated CRISPR-Cas9 mutagenesis is independent of the ku80 pathway, indicating that this system can function as a powerful and versatile genome-editing tool in clinical Aspergillus isolates.

KEYWORDS:

CRISPR mutagenesis; In situ tag-insertion; Microhomology; Multi-locus genomic mutagenesis

PMID:
26701308
DOI:
10.1016/j.fgb.2015.12.007
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center