CRISPR Editing of the gene FST4 in an Industrial Fungus used in Commercial Biomaterials

This study utilizes CRISPR-Cas9 technology with the goal of improving the structural characteristics of an industrial strain of fungus used by Ecovative Design LLC in the production of commercial biomaterials. These biomaterials are sustainable, renewable, and ideally will replace plastics like Styrofoam in all packaging materials. The gene of interest for our genetic manipulations with CRISPR-Cas9 system, FST4, has been shown by others in a related fungus to be essential in mushroom formation. This undesired reproductive growth habit would interfere with the long-term structure of the biomaterials and also their function as a packaging material. Our approach was to use CRISPR technology to disable (knockout) the FST4 gene, in order to prevent mushroom formation. Gibson Assembly was used to create three different FST4 guide RNA CRISPR constructs, which were then integrated into the industrial fungus via  Agrobacterium-mediated transformation. The correct sequences of each recombinant construct were verified via restriction enzyme digest polymerase chain reaction, and also DNA sequencing. Gene editing experiments using these constructs are in progress, and results will be discussed.