Title

CRISPR Editing Of The Gene FST4 In An Industrial Fungus Used In Commercial Biomaterials

Date of Award

6-2019

Document Type

Restricted (Opt-Out)

Degree Name

Bachelor of Science

Department

Neuroscience

First Advisor

J. Stephen Horton

Keywords

CRISPR, fungus, fruiting body, mushrooms, ATMT, genetic engineering

Abstract

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. FTS4 is the gene of interest for our genetic manipulations with CRISPR-Cas9 system, and has been shown previously 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 tumefaciens mediated transformation. The correct sequences of each recombinant construct were verified via polymerase chain reaction and DNA sequencing. One of the ten tested fungal transformants contained mutations in the FST4 gene in the region characteristic of CRISPR editing. However, transformants sharing the same phenotype as the mutated transformant were found to contain an unaltered wild type FST4 allele. Thus, we could not conclude that the transformant’s abberrant phenotype was due to any particular mutation in FST4. We are in the process of repeating the experiment with a new vector backbone substituting an RNA Pol II promoter for the tRNA Pol III promoter present in our original constructs.

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