Ganoderma is a genus of wood decaying fungi, some members of which are cultivated to produce mushrooms for medicinal purposes. In both plants and fungi, the hormone ethylene has been shown to regulate aspects of growth and development, especially in response to environmental stressors. ETR1 and ETR2 are two known ethylene-signaling receptors in mushroom-producing fungi, and the knockdown of expression via RNAi of both these two genes has been accompanied by reduced sensitivity to ethylene inhibition in the button mushroom Agaricus bisporus. Reduced ethylene inhibition has increased the rate of mycelial growth in Agaricus, and in a similar vein, it has been found that a knockout in ETR1 of Ganoderma results in a modest increase in growth rate. The goal of this project is to investigate whether a double knockout in both ETR1 and ETR2 would result in an even greater increase in growth rate of the resulting "double null" mutant. In order to create this double mutant, we are employing CRISPR technology via introduction of transient Cas9/guide RNA RiboNucleoProtein (RNP) complexes to inactivate both ETR1 and ETR2 simultaneously. To enable this, a "donor DNA" recombinant DNA construct for ETR2 has been developed, and will be utilized for the purposes of Homology-Directed Repair (HDR) in our CRISPR experiments. The double knockout will be compared to the existing ETR1 single knockout, and analyzed under a variety of conditions to determine if there is a synergistic effect on the growth of the resulting mutant strain of Ganoderma.
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