Grant: Discovery Grants Program - Individual

BACKGROUND and PROGRESS. Eukaryotic microalgae are a diverse group of ecologically significant organisms that are emerging as synthetic biology platforms. One barrier to genetic manipulation of microalgae are robustly active gene-editing tools. In the past funding period, my research program embarked on a new direction and developed plasmid-based clustered regularly interspaced palindromic repeat (CRISPR) nucleases for gene editing in Phaeodactylum tricornutum and generated gene-knockouts in biosynthetic pathway genes for plasmid-based complementation and biocontainment. We used Oxford Nanopore sequencing to complete a telomere-to-telomere genome assembly and to demonstrate that CRISPR-editing events resulted in loss of heterozygosity. We constructed a fluorescent-based traffic-light reporter system to track gene-editing outcomes and showed that this system can detect CRISPR editing. One conclusion from these investigations is that we do not yet understand how CRISPR-induced breaks are processed by DNA repair pathways in P. tricornutum. RESEARCH OBJECTIVES. The long-term goal of my research program is to develop tools for robust and predictable genome editing to enhance P. tricornutum synthetic biology. The objective of the next 5-year Discovery Grant is to understand factors that influence gene-editing outcomes in P. tricornutum. This will be accomplished by three objectives. 1. To characterize editing outcomes using a traffic-light-reporter system that will allow us to distinguish the ratio of non-homologous end joining versus homology-directed DNA repair events. We will also use this system to test if regulating CRISPR expression and the types of DNA ends generated at cleavage influences repair outcomes. 2. To identify and characterize NHEJ functions by using proximity-dependent biotin identification to identify Ku70 interacting partners. We will use CRISPR editing to knockout or make mutations in candidate genes and determine the effect on DNA repair outcomes. 3. To enhance the rate of targeted integration of foreign DNA by testing the hypothesis that our TevCas9 dual nuclease will limit non-productive editing and integration events by preventing target site regeneration. IMPACT. Gene editing of diatom genomes is a recent development but suffers from a lack of knowledge about functional diatom DNA repair pathways. Our research will lead to a greater understanding of repair of nuclease-induced double-strand breaks that will help develop strategies to bias repair toward desired outcomes. We anticipate that these tools and strategies will provide a valuable resource to the diatom research community. Given the recent interest in P. tricornutum as a 'diatom cell factory', these tools would also enhance synthetic biology applications. The hypothesis-driven research proposed in this application offers excellent training opportunities for HQP at different career stages.