The Construction Of CRISPR/Cas9-based Cdk5 Knockout System For Trichoderma Atroviride

Plant disease problems seriously affect the use of natural resources in agriculture.At present,people mainly use chemical pesticides to control plant pathogens,but the misuse of chemical pesticides can cause environmental pollution and lead to resistance of pathogens to chemical pesticides,causing harm to human and animal health and other aspects.Biological control can be an effective solution to these problems.Trichoderma atroviride is an important biocontrol bacterium that can suppress a variety of plant pathogens,but the molecular mechanism by which Trichoderma atroviride exerts its biocontrol function is not yet fully understood.The function of the cdk5 gene has rarely been reported.CRISPR/Cas9 was found to be superior as a novel gene editing technology with easy operation,low cost,high efficiency,and speed.However,its application in fungi is limited by the low homologous recombination rate and the lack of efficient genetic transformation systems,promoters,and effective screening markers.The main focus of this study was to construct a gene knockout system for the Trichoderma atroviride cdk5 gene using CRISPR/Cas9 gene editing technology.In this paper,we used bioinformatics analysis to show that CDK5 is widespread and highly conserved in Trichoderma atroviride;using CRISPR/Cas9 gene editing,we constructed a knockout system in Trichoderma atroviride and failed to obtain the correct transformants for the knockout of cdk5.In order to exclude the possibility of negative results due to the ineffectiveness of the constructed knockout system,we conducted a validity test of the constructed knockout system,targeting the ura5 gene,and successfully achieved the targeted mutation of ura5 by using two strategies: in vitro assembled Cas9/g RNA complex and Trichoderma atroviride 5S r RNA:sg RNA,and obtained a high knockout efficiency.A high knockdown efficiency was achieved.In this paper,we applied CRISPR/Cas9 system in Trichoderma atroviride for gene knockout practice,which eliminated the obstacles for the subsequent study of cdk5 gene function and provided the theoretical basis for the screening and validation of Trichoderma atroviride functional genes and the construction of engineering strains.