A simple CRISPR/Cas9 system for efficiently targeting genes of Aspergillus section Flavi species, Aspergillus nidulans, Aspergillus fumigatus, Aspergillus terreus and Aspergillus niger

Authors: Chang, Perng Kuang

Submitted to: Microbiology Spectrum
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/23/2022
Publication Date: 1/18/2023
Citation: Chang, P.-K. 2023. A simple CRISPR/Cas9 system for efficiently targeting genes of Aspergillus section Flavi species, Aspergillus nidulans, Aspergillus fumigatus, Aspergillus terreus and Aspergillus niger. Microbiology Spectrum. 11(1):1-18. https://doi.org/10.1128/spectrum.04648-22.
DOI: https://doi.org/10.1128/spectrum.04648-22

Interpretive Summary: Aspergillus flavus is a pathogen of economically important crops and produces hepatocarcinogenic aflatoxins. It is also an opportunistic human pathogen, which is the second leading causative agent of invasive and non-invasive aspergillosis. Significant economic losses have resulted from aflatoxin contamination of food and feed. Vast genomic resources for A. flavus and other aflatoxin-producing aspergilli play an important role in our understanding of toxin production and fungus-plant interactions. In this work, an improved, more efficient gene-targeting system than current ones for Aspergillus functional genomics studies was developed. Its efficacy was demonstrated in different morphotypes of A. flavus and in several species belong to the same group (section Flavi) as well as in genetically distant aspergilli. The developed system will facilitate the elucidation of gene functions, which would lead to better strategies for reducing aflatoxin contamination of agricultural commodities.

Technical Abstract: CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease) genome-editing systems have been developed for many filamentous fungi. Reported gene-targeting efficiencies vary greatly and are dependent on delivery methods, repair mechanisms of induced double-stranded breaks, selection markers, and genetic backgrounds of transformation recipient strains. For Aspergillus flavus, a pathogen of considerable economic and health concern, successful gene knockout work for more than a decade relies nearly exclusively on using nonhomologous end-joining pathway (NHEJ)-deficient recipients via forced double-crossover recombination of homologous sequences. In this study, a simple CRISPR/Cas9 system that gave extremely high (>95 %) gene-targeting frequencies in A. flavus was developed. It contained a shortened Aspergillus nidulans AMA1 autonomously replicating sequence that maintained good transformation frequencies and Aspergillus oryzae ptrA as the selection marker for pyrithiamine resistance. Expression of codon-optimized cas9 gene was driven by A nidulans gpdA promoter and trpC terminator. Expression of single guide RNA (sgRNA) cassettes was controlled by A. flavus U6 promoter and terminator. The high transformation and gene-targeting frequencies of this system made generation of A. flavus gene knockouts with or without phenotypic changes effortlessly. Additionally, it allowed knocking out multiple genes efficiently either by sequential transformation or co-transformation of individual sgRNA vectors if desired. For example, triple- gene knockouts of A. flavus conidial pigment genes (olgA/copT/wA or olgA/yA/wA) were quickly generated in a month by the sequential approach. Co-transforming sgRNA vectors targeting A. flavus kojA, yA, and wA gave 52%, 40%, and 8% of single-, double-, and triple-gene knockouts, respectively. The system was readily appliable to other section Flavi aspergilli (A. parasiticus, A. oryzae, A. sojae, A. nomius, A. bombycis and A. pseudotamarii) with comparable transformation and gene-targeting efficiencies. Moreover, it gave satisfactory gene-targeting efficiencies (> 90%) in A. nidulans (section Nidulantes), A. fumigatus (section Fumigati), A. terreus (section Terrei), and A. niger (section Nigri). It likely will have a broad application in aspergilli. This cross-Aspergillus section, highly efficient system is for wild-type isolates and does not require homologous donor DNAs to be added or NHEJ-deficient strains to be created or forced recycling of knockout recipients to be performed for multiple-gene targeting. Hence, it simplifies and expedites the gene-targeting process significantly.