The purpose of our project is to utilize the a filamentous-fungi-specific CRISPR-Cas9 system 1 in conjunction with an optogenetic system 2 to construct a fast acting kill switch system for genetically engineered fungi, which is becoming ever more popular method for dealing with insect pest around the world. Two plasmids will be constructed, each carrying a module of our two part system. Metarhizium anisopliae ARSEF 549 will be transformed with both plasmids and inoculated on live specimen of Bactrocera dorsalis and Spodoptera litura to test the viability and efficacy of the system. If the system is viable, we think that it could be adapted for other organism for similar purposes.
Team:NYMU-Taipei/Notebook-Protocol
Protocol
Project Description
The system will be divided into two modules:
1. Optogenetic activation module:
The main working components of this module is the VP-EL222 gene and the Pmcl1 promoter. Both components will be introduced to M. anisopliae on the plasmid pBARGPE1. VP-EL222 is a protein that contains a blue-light inducible LOV domain and a transcriptional activator domain responsible for the activation of the execution module. Pmcl1 is a hemolymph-induced promoter from M. anisopliae that activates when the mycelium enters the hemolymph of its host. Limiting the production of the VP-EL222 protein, which will be under the control of Pmcl1, to only after the fungus has successfully penetrated the hosts’ cuticle. This allows the fungi to proliferate in the darkness of the hemolymph. However, when the fungi reaches the end of its life cycle, it must penetrate the insect’s cuticle from the inside to produce conidia on the surface of the host’s cuticle, which puts the mycelium in contact with sunlight. This is when VP-EL222 proteins will dimerize and activate the CRISPR-Cas9 execution module.
2. CRISPR-Cas9 execution module:
The function of this module is to target several essential genes to reduce the survivability and the competiveness of our GM fungi. These target genes will serve as templates for sgRNA, allowing Cas9 to mutate parts of the genes’ sequences to achieve gene disruption. Currently, we are targeting two genes, MrPHR1 and MrPHR2, which encodes photolysase M. anisopliae. The disruption of these genes will reduce the UV resistance and disrupt the trehalose synthesis of our target fungi 3, thus reducing its survivability. We will also be searching for other genes that are essential to M. ansiopliae’s survival to further reduce our fungi’s survivability.
After transforming M. anisopliae, we will be inoculating that carries the kill switch system onto two species of insect, Bactrocera dorsalis and Spodoptera litura, to ensure that the whole system functions as intended.
