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具有重要生态意义的生物体，它们正在成为合成生物学平台，而强大的基因编辑工具是阻碍其发展的一个障碍。方向并开发了基于质粒的成簇规则间隔回文重复（CRISPR）核酸酶，用于三角褐指藻的基因编辑，并生成我们使用牛津纳米孔测序完成了端粒到端粒基因组的组装，并证明了 CRISPR 编辑事件导致了杂合性的丧失。红绿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。这些研究的一个结论是，我们尚不了解 CRISPR 是如何诱导的。研究目标 我的研究计划的长期目标是开发强大且可预测的基因组编辑工具，以增强三角藻的合成生物学。发现补助金旨在了解影响三角角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，这将使我们能够区分基因编辑结果。非同源末端连接与同源定向 DNA 修复事件的比率 我们还将使用该系统来测试调节 CRISPR 表达和切割时生成的 DNA 末端类型是否会影响修复结果。邻近依赖性生物素鉴定，以识别 Ku70 相互作用伙伴。我们将使用 CRISPR 敲除或突变候选基因，并确定对 DNA 修复结果的影响 3. 通过以下方式提高外源 DNA 的靶向整合率。测试我们的 TevCas9 双核酸酶将通过阻止靶位点再生来限制非生产性编辑和整合事件的假设是最近的一项进展，但我们的研究缺乏对功能性硅藻 DNA 修复途径的了解。将导致对核酸酶诱导的双链断裂的修复有更深入的了解，这将有助于制定使修复偏向于预期结果的策略。鉴于最近人们对三角角藻作为“硅藻细胞工厂”的兴趣，这些工具也将增强合成生物学应用，该应用中提出的假设驱动研究为不同职业阶段的 HQP 提供了极好的培训机会。