Understanding and manipulating gene-editing outcomes in eukaryotic microalgae

了解和操纵真核微藻的基因编辑结果

• 批准号: RGPIN-2022-05459
• 负责人: Edgell, David
• 金额: $ 2.91万
• 依托单位: University of Western Ontario
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744073
• 关键词: Understanding; manipulating; gene; editing; outcomes

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.

背景和进展。真核微藻是一类多样性的具有生态意义的生物，它们正在成为合成生物学平台。微藻基因操作的一个障碍是非常活跃的基因编辑工具。在过去的资助期间，我的研究项目走上了一个新的方向，开发了基于质粒的聚集性规则间隔回文重复(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。我们使用牛津纳米孔测序来完成端粒到端粒基因组的组装，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，我们还不知道CRISPR诱导的断裂是如何通过DNA修复途径在三角帆船中进行处理的。研究目标。我的研究计划的长期目标是开发用于强大和可预测的基因组编辑的工具，以增强三角葡萄球菌的合成生物学。下一项为期5年的发现基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.使用红绿灯报告系统来表征编辑结果，该系统将允许我们区分非同源末端连接事件与同源定向DNA修复事件的比率。我们还将使用这个系统来测试调控CRISPR的表达和在切割时产生的DNA末端类型是否影响修复结果。2.通过使用邻近依赖的生物素鉴定来鉴定Ku70相互作用伙伴，从而鉴定和鉴定NHEJ的功能。我们将使用CRISPR编辑来敲除或使候选基因发生突变，并确定对DNA修复结果的影响。3.通过测试我们的TevCas9双核酸酶将通过阻止靶部位再生来限制非生产性编辑和整合事件的假设，以提高外源DNA的靶向整合速度。冲击力。硅藻基因组的基因编辑是最近的发展，但缺乏对硅藻DNA修复途径的了解。我们的研究将有助于更好地理解核酸酶诱导的双链断裂的修复，这将有助于制定策略，使修复偏向预期的结果。我们预计，这些工具和战略将为硅藻研究界提供宝贵的资源。考虑到最近人们对三角褐指藻作为“硅藻细胞工厂”的兴趣，这些工具也将加强合成生物学的应用。本申请中提出的假设驱动研究为HQP在不同职业阶段提供了极好的培训机会。