NSF Postdoctoral Fellowship in Biology FY 2021: Continuous Directed Evolution and CRISPR-Cas9 as a Combinatorial Approach to Engineering Plant Metabolism

该行动为2021财年的NSF植物基因组博士后生物学研究奖学金提供资金。该研究金支持研究员在东道实验室的研究和培训计划，研究员还提出了扩大生物学参与的计划。Bryan Leong博士的研究和培训计划的标题是“持续定向进化和CRISPR-Cas9作为工程植物代谢的组合方法”。该奖学金的主办机构是佛罗里达大学，赞助科学家是安德鲁·汉森博士。植物是当今社会的主力，提供食物、纤维、燃料、药物等。人类长期以来一直为此目的培育植物，但传统的育种有各种局限性。它通常依赖于从野生物种中获取的性状或诱变来实现预期的结果。使用当前的方法获得新功能可能很困难，但最近的进展使其变得更加可行。更好的植物酶可以通过持续的定向进化来开发，这涉及将酶插入微生物中，并将微生物生长与酶的改进功能相结合。具有更好酶的微生物生长更快，从而能够发现新的酶变体，例如，抗除草剂或可以增强有价值化学物质的积累。CRISPR-Cas9允许编辑植物基因组而无需持久的转基因。这种编辑过程可以将改进的酶变体返回到植物中用于下游应用。该项目是通过连续定向进化和CRISPR-Cas9开发更好的酶功能的联合收割机的原理证明。这两项技术将彻底改变农业。培训目标包括发展合成生物学专业知识，以补充现有技能，积极参与向公众的科学宣传，并提高沟通技能。更广泛的影响包括帮助在佛罗里达大学组织“迷恋植物日”，并志愿参加“科学家在每个佛罗里达学校”项目，向学生介绍他们社区正在进行的研究。虽然人类已经通过各种方法对植物进行了遗传改良，但通过代谢工程进行进一步改良仍有巨大的潜力。然而，在这种工程中使用转基因植物会带来耻辱。合成生物学工具，如连续定向进化和CRISPR-Cas9，正在开辟植物代谢工程的新领域。酶适应度景观可以使用连续定向进化来快速探索，以连续合并多个有益的突变。CRISPR-Cas9彻底改变了基因组编辑，并带来了强大的新技术，如TargetAID，可以精确编辑特定基因中的碱基对。该项目将利用CRISPR-Cas9在拟南芥中的连续定向进化和无缝编辑的能力，以原理验证的组合方法进行。连续定向进化将用于在微生物中进化拟南芥酶中的除草剂抗性，然后使用TargetAID和CRISPR-Cas9将这些抗性突变引入拟南芥中，而没有持久的转基因盒。这种结合的方法，然后将被应用到工程反馈不敏感到拟南芥氨基酸生物合成酶。在这个项目中产生的数据将被提交到适当的公共存储库，结果将尽可能在开放获取期刊上发表。这个奖项反映了NSF的法定使命，并被认为是值得通过使用基金会的知识价值和更广泛的影响审查标准进行评估的支持。

This action funds an NSF Plant Genome Postdoctoral Research Fellowship in Biology for FY 2021. The fellowship supports a research and training plan in a host laboratory for the Fellow who also presents a plan to broaden participation in biology. The title of the research and training plan for this fellowship to Dr. Bryan Leong is “Continuous Directed Evolution and CRISPR-Cas9 as a Combinatorial Approach to Engineering Plant Metabolism”. The host institution for the fellowship is the University of Florida and the sponsoring scientist is Dr. Andrew Hanson.Plants are workhorses of today’s society that provide food, fiber, fuel, medicines, and more. Humans have long bred plants for such purposes, but traditional breeding has various limitations. It often relies on traits taken from wild species or on mutagenesis to achieve the desired outcomes. Obtaining new functions can be difficult using current approaches, but recent advances have made this more feasible. Better plant enzymes can be developed by continuous directed evolution, which involves inserting the enzyme into a microbe and coupling microbial growth to improved function of the enzyme. The microorganism with the better enzyme grows faster, enabling discovery of new enzyme variants that are, for example, herbicide-resistant or that can enhance accumulation of valuable chemicals. CRISPR-Cas9 allows editing of plant genomes without lasting transgenes. This editing process can return the improved enzyme variants to plants for downstream applications. This project is a proof-of-principle to combine the development of better enzyme function through continuous directed evolution and CRISPR-Cas9. These two technologies stand to revolutionize agriculture. The training objectives include developing expertise in synthetic biology to complement existing skillsets, actively participating in scientific outreach to the public, and refining communication skills. Broader impacts include helping to organize Fascination with Plants day at the University of Florida and volunteering for the Scientist in Every Florida School program to introduce students to ongoing research in their community.Humans use plants for food, fiber, fuel, medicines, industrial chemicals, and other purposes. While humans have genetically improved plants by various methods, there is still enormous potential for further improvement by metabolic engineering. Using transgenic plants in this engineering carries a stigma, however. Synthetic biology tools like continuous directed evolution and CRISPR-Cas9 are now opening new frontiers in plant metabolic engineering. Enzyme fitness landscapes can be quickly explored using continuous directed evolution to incorporate multiple beneficial mutations in succession. CRISPR-Cas9 has revolutionized genome editing and led to powerful new technologies like TargetAID that can precisely edit base pairs in specific genes. This project will use the power of continuous directed evolution and seamless editing by CRISPR-Cas9 in Arabidopsis in a proof-of-principle combined approach. Continuous directed evolution will be used to evolve herbicide resistance in Arabidopsis enzymes in microbes, followed by introduction of those resistance mutations into Arabidopsis using TargetAID and CRISPR-Cas9 with no lasting transgenic cassettes. This combined approach will then be applied to engineer feedback-insensitivity into Arabidopsis amino acid biosynthesis enzymes. Data generated in this project will be submitted to the appropriate public repositories and results will be published in open access journals whenever possible.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.