CRISPR/Cas9-Based Genome Editing and Its Application in Aspergillus Species

Abstract

Aspergillus, a genus of filamentous fungi, is extensively distributed in nature and plays crucial roles in the decomposition of organic materials as an important environmental microorganism as well as in the traditional fermentation and food processing industries. Furthermore, due to their strong potential to secrete a large variety of hydrolytic enzymes and other natural products by manipulating gene expression and/or introducing new biosynthetic pathways, several Aspergillus species have been widely exploited as microbial cell factories. In recent years, with the development of next-generation genome sequencing technology and genetic engineering methods, the production and utilization of various homo-/heterologous-proteins and natural products in Aspergillus species have been well studied. As a newly developed genome editing technology, the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has been used to edit and modify genes in Aspergilli. So far, the CRISPR/Cas9-based approach has been widely employed to improve the efficiency of gene modification in the strain type Aspergillus nidulans and other industrially important and pathogenic Aspergillus species, including Aspergillus oryzae, Aspergillus niger, and Aspergillus fumigatus. This review highlights the current development of CRISPR/Cas9-based genome editing technology and its application in basic research and the production of recombination proteins and natural products in the Aspergillus species.

Establishment of CRISPR/Cas9 Genome-Editing System Based on Dual sgRNAs in Flammulina filiformis

“…CRISPR/Cas9-mediated genome editing is a revolutionary technology that has been widely applied in filamentous fungi, including Basidiomycetes [ 11 , 12 , 13 ]. However, due to the limited genetic manipulation and transformation methods, there have been relatively few reports describing successful CRISPR systems in edible fungi.…”

A Simple CRISPR/Cas9 System for Efficiently Targeting Genes of Aspergillus Section Flavi Species, Aspergillus nidulans, Aspergillus fumigatus, Aspergillus terreus, and Aspergillus niger

“…The developed system should promote and facilitate gene function studies in S-type A. flavus . Its gene-targeting frequencies in A. flavus , conservatively, reached over 95%, and roughly 80% of knockouts would contain small deletions that can be easily determined ( Table 3 ); these targeting frequencies were better than those reported for NHEJ-deficient systems that rely on homology-directed repair ( 18 ). This improved CRISPR/Cas9 technology thus could make the dependence of over a decade on A. flavus NHEJ-deficient recipient strains and auxotrophic selection markers a thing of the past.…”

“…To increase the efficiencies, homologous donor DNAs (plasmids or DNA fragments) often have to be included, especially when NHEJ-deficient recipient strains are employed. A very recent review gives an excellent summary of delivery methods, DNA repair mechanisms, and targeting efficiencies on established CRISPR/Cas9 systems for aspergilli ( 18 ). The lack of a simple, efficient, and universal system means current CRISPR/Cas9 technology for aspergilli still had much room for improvement.…”

Putative Core Transcription Factors Affecting Virulence in Aspergillus flavus during Infection of Maize

“…Gene knockout was performed using the established CRISPR/Cas9 technology [ 19 ]. DNA fragments that encoded single guide RNAs for targeting genes of interest were PCR amplified using pAf-CRISPR-yA (Addgene plasmid #191015) as the template.…”