Open AccessHigh‐Throughput Gene Replacement in Aspergillus fumigatus
Author(s) -
Zhao Can,
Fraczek Marcin G.,
Dineen Lauren,
Lebedinec Ressa,
Macheleidt Juliane,
Heinekamp Thorsten,
Delneri Daniela,
Bowyer Paul,
Brakhage Axel A.,
Bromley Michael
Publication year - 2019
Publication title -
current protocols in microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.813
H-Index - 35
eISSN - 1934-8533
pISSN - 1934-8525
DOI - 10.1002/cpmc.88
Subject(s) - aspergillus fumigatus , biology , mutant , computational biology , gene , functional genomics , aspergillosis , gene knockout , fungal pathogen , transformation (genetics) , microbiology and biotechnology , genomics , genetics , genome , immunology
Aspergillus fumigatus is a human pathogen and the principal etiologic agent of invasive and chronic aspergillosis leading to several hundreds of thousands of deaths every year. Very few antifungals are available to treat infections caused by A. fumigatus , and resistance is developing to those we have. Our understanding of the molecular mechanisms that drive pathogenicity and drug resistance have been hampered by the lack of large mutant collections, which limits our ability to perform functional genomics analysis. Here we present a high‐throughput gene knockout method that combines a highly reproducible fusion PCR method to enable generation of gene replacement cassettes with a multiwell format transformation procedure. This process can be used to generate 96 null mutants within 5 days by a single person at a cost of less than £18 ($24) per mutant and is being employed in our laboratory to generate a barcoded genome‐wide knockout library in A. fumigatus . © 2019 The Authors.