Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy [to the] School of Applied Systems Biology, Faculty of Science, Technology and Engineering, La Trobe University, Victoria, Australia.
Genome editing technology is a powerful tool for improving agricultural productivity and end-product quality. Epichloë endophytes of pasture grasses can be modified using genome editing to eliminate animal toxicity, while retaining the benefits of enhanced pasture persistence. This thesis investigates the development and application of EXZACTTM Delete (i.e. loss-of-function) genome editing technology using zinc finger nucleases (ZFNs) in Epichloë. First, DNA doublestranded break (DSB) repair pathways in Epichloë species representing E. festucae, E. festucae var. lolii, LpTG-3, and LpTG-4 were identified in silico. Whole genome and RNA-seq data was used to identify and verify in silico endogenous DSB repair pathways that can be utilized for genome editing in Epichloë. An optimized PEG-mediated transfection protocol for Epichloë was then developed and implemented. This protocol permits molecular analysis of protoplasts 24 hours post-transfection without the time and labor consuming steps of regenerating protoplasts. Additionally, gain-of-function and loss-of-function of the sgfp gene was quantitatively analyzed using flow cytometry analysis (FCA). The high transfection efficiency of the optimized protocol may be due, in part, to high transient expression rates and is useful for avoiding antibiotic resistance selection. Finally, insertion/deletion (indel) detection protocols in Epichloë were designed and implemented to quantify ZFN-mediated deletion events. In this study, the frequency of indel events, relying on the endogenous DSB repair pathway, was low. Amplicon sequencing technology was applied to successfully observe indels at a frequency of 0.005 percent (1 indel for every 20000 reads). Droplet-digital PCR showed promise in detecting indels at higher frequencies (0.13 percent) and FCA proved to be the least sensitive method. Addressing the low number ZFN-mediated deletion events such as efficient DSB repair machinery and vector design would be the next step in these studies. The results obtained from these studies provide the basis for genome editing in Epichloë and for creating the perfect perennial ryegrass endophyte with low or no regulatory burden.
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