Abstract
Beauveria bassiana is an environmentally friendly alternative to chemical insecticides against various agricultural insect pests and vectors of human diseases. However, its application has been limited due to slow kill and sensitivity to abiotic stresses. Understanding of the molecular pathogenesis and physiological characteristics would facilitate improvement of the fungal performance. Loss-of-function mutagenesis is the most powerful tool to characterize gene functions, but it is hampered by the low rate of homologous recombination and the limited availability of selectable markers. Here, by combining the use of uridine auxotrophy as recipient and donor DNAs harboring auxotrophic complementation gene ura5 as a selectable marker with the blastospore-based transformation system, we established a highly efficient, low false-positive background and cost-effective CRISPR/Cas9-mediated gene editing system in B. bassiana. This system has been demonstrated as a simple and powerful tool for targeted gene knock-out and/or knock-in in B. bassiana in a single gene disruption. We further demonstrated that our system allows simultaneous disruption of multiple genes via homology-directed repair in a single transformation. This technology will allow us to study functionally redundant genes and holds significant potential to greatly accelerate functional genomics studies of B. bassiana.
Highlights
Beauveria bassiana is an environmentally friendly alternative to chemical insecticides against various agricultural insect pests and vectors of human diseases
Our study demonstrated that the CRISPR/Cas[9] system showed very high frequency of homologous recombination when a pair of 250 bp flanking homology arms were used
Gene disruption or replacement has relied on the use of antibiotic or herbicide resistance markers
Summary
Beauveria bassiana is an environmentally friendly alternative to chemical insecticides against various agricultural insect pests and vectors of human diseases. Genetic engineering has been proved to be a promising strategy to significantly improve fungal virulence, increase stress tolerance, and reduce transmission of mosquito-borne diseases[10,11,12] This highlights the need for a better understanding of virulence determinants and physiological characteristics in B. bassiana. The Agrobacterium-mediated gene targeting method is technically complicated, laborious, together with low rates of homologous recombination and we usually need to screen hundreds to thousands of transformants across multiple transformation attempts to obtain the desired mutant[18] Another limitation for mutagenesis in B. bassiana is the lack of adequate dominant selectable markers. The mutants required exogenous uridine for growth and were positively selected in the medium containing 5-fluoroorotic acid (5-FOA)[25], indicating that uridine auxotrophy can be used as a selectable marker for the transformation of B. bassiana
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