CRISPR/Cas9-Mediated Multiplexed Genome Editing in Aspergillus oryzae

Abstract

Aspergillus oryzae has great potential and competitive advantages to be developed as an excellent expression system, owing to its powerful protein secretion ability, complex post-translational modification, and safety characteristics. However, the low efficiency of genetic modification and gene function analysis is an urgent problem to be solved in A. oryzae and other filamentous fungal systems. Therefore, establishing efficient genetic transformation and multiplexed genome editing tools is significant for developing A. oryzae expression systems, and revealing its intrinsic mechanisms. In this study, the high-efficiency transformation of A. oryzae was achieved by optimizing the preparation conditions of protoplasts, and the random editing efficiency of the CRISPR/Cas9 system in A. oryzae for single and double genes reached 37.6% and 19.8%, respectively. With the aid of the selection marker, such as color or resistance, the editing efficiency of single and double genes can reach 100%. Based on the developed CRISPR/Cas9 genome editing method, the heterologous lipase gene (TLL) achieves precise integration at different genetic loci in one step. The efficient and accurate acquisition of positive transformants indicated that the morphological gene yA could be used as a helpful selection marker for genome editing in A. oryzae. In conclusion, the developed system improves the efficiency of transformation and multiplexed genome editing for A. oryzae. It provides a practical method for developing the A. oryzae high-efficiency expression system for heterologous proteins.

High-throughput droplet microfluidics screening and genome sequencing analysis for improved amylase-producing Aspergillus oryzae

“…Trimmomatic was used for data processing. The target gene selected after analysis was edited and quickly screened using the CRISPR–Cas9 system based on the morphological gene yA as an indicator [ 44 ]. Mutants were selected by A. oryzae colony PCR and sequencing for subsequent experimental verification [ 44 ].…”

“…The target gene selected after analysis was edited and quickly screened using the CRISPR–Cas9 system based on the morphological gene yA as an indicator [ 44 ]. Mutants were selected by A. oryzae colony PCR and sequencing for subsequent experimental verification [ 44 ]. The primers and plasmids required for the construction of the CRISPR–Cas9 system are listed in Additional file 1 : Tables S1 and S2.…”

Industrial applications of fungal lipases: a review

“…Similarly, in this way, we can engineer the genomes (by adding high-potential lipase genes) of lipase-producing fungi with the CRISPR/Cas9 system to increase the lipase production ability of these fungi. Li et al (2023) modified the genome of Aspergillus oryzae with CRISPR/Cas9 technology, and the heterologous lipase gene (TLL) achieved the precise integration of different genetic loci in one step.…”

Establishment, optimization, and application of genetic technology in Aspergillus spp.

“…Moreover, multiple sgRNA expression plasmids can be transferred into one cell to edit multiple genes synchronous, such as A . oryzae ( Li et al, 2023 ) and even have the potential function of genome screening ( Cong et al, 2013 ; Zetsche et al, 2017 ; Zeng et al, 2022 ). At present, a considerable part of common Aspergillus gene function studies have applied the CRISPR technology, also have applied in fungal factories, such as glucoamylase-hyperproducing industrial in A .…”