Understanding and manipulating gene-editing outcomes in eukaryotic microalgae

背景与进展。真核微藻是一组多样的具有重要生态意义的生物体，正在成为合成生物学平台。微藻遗传操作的一个障碍是强大的活性基因编辑工具。在过去的资助期间，我的研究计划开始了一个新的方向，并开发了基于质粒的成簇规则间隔回文重复序列（CRISPR）核酸酶，用于三角褐指藻的基因编辑，并在生物合成途径基因中产生基因敲除，用于基于质粒的互补和生物遏制。我们使用Oxford Nanopore测序来完成端粒到端粒的基因组组装，并证明CRISPR编辑事件导致杂合性丢失。我们构建了一个基于荧光的交通灯报告系统来跟踪基因编辑的结果，并表明该系统可以检测CRISPR编辑。从这些研究中得出的一个结论是，我们还不了解CRISPR诱导的断裂如何通过三角褐指藻中的DNA修复途径进行处理。研究替代品。我的研究计划的长期目标是开发强大和可预测的基因组编辑工具，以增强三角褐指藻合成生物学。下一个5年发现补助金的目标是了解影响三角褐指藻基因编辑结果的因素。这将通过三个目标来实现。 1.使用交通灯报告系统表征编辑结果，该系统将使我们能够区分非同源末端连接与同源指导的DNA修复事件的比率。我们还将使用该系统来测试调控CRISPR表达和切割时产生的DNA末端类型是否会影响修复结果。 2.通过使用邻近依赖性生物素鉴定来鉴定Ku70相互作用伴侣，以鉴定和表征NHEJ功能。我们将使用CRISPR编辑来敲除或突变候选基因，并确定对DNA修复结果的影响。 3.为了通过测试我们的TevCas9双核酸酶将通过防止靶位点再生来限制非生产性编辑和整合事件的假设来提高外源DNA的靶向整合速率。冲击硅藻基因组的基因编辑是最近的发展，但缺乏对功能性硅藻DNA修复途径的了解。我们的研究将导致对核酸酶诱导的双链断裂的修复有更深入的了解，这将有助于制定策略，使修复偏向预期的结果。我们期望这些工具和策略将为硅藻研究界提供宝贵的资源。鉴于最近对三角褐指藻作为“硅藻细胞工厂”的兴趣，这些工具也将增强合成生物学的应用。本申请提出的假设驱动研究为不同职业阶段的HQP提供了良好的培训机会。

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.