High-Throughput Gene Replacement in Aspergillus fumigatus

Abstract

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.

Keywords: Aspergillus fumigatus; fusion PCR; genome-wide knockout library; high-throughput gene editing.

Figure 1

Schematic overview of fusion PCR–based generation of gene knockout mutants in A. fumigatus. (A) Upstream and downstream fragments are amplified with primers P1 and P2 and with P3 and P4. The hph selective marker cassette is amplified with primers hph_F and hph_R. Primers P2 and P3 are designed to introduce generic linkers (shown in yellow and navy) that would allow fusion to the central marker cassette. (B) The upstream and downstream fragments are fused to the hph selective cassette by fusion PCR using nested primers (P5 and P6) creating a linear fragment suitable for transformation. (C) Replacement of a hypothetical target gene by hph selective cassette during transformation.

Figure 2

Typical results from agarose gel electrophoresis of 96‐well formatted PCR amplifications. (A) Amplified upstream fragments. Each product should exhibit a single band at around 1.2 kb. (B) Amplified downstream fragments. Each product should exhibit a single band at around 1.2 kb. (C) Amplified fusion PCR fragments. Each product should exhibit a major band at around 4.8 kb.

Figure 3

Overview of steps for a 96‐well formatted transformation.