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

这一行动为2021财年NSF植物基因组生物学博士后研究奖学金提供了资金。该奖学金支持在东道主实验室为该研究员制定的研究和培训计划，该研究员还提出了扩大生物学参与度的计划。Bryan Leong博士的这项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、药品等。人类为此目的培育植物由来已久，但传统的育种有各种局限性。它通常依靠从野生物种中获得的特征或通过突变来实现预期的结果。使用目前的方法很难获得新的功能，但最近的进展使这一点变得更加可行。通过持续的定向进化可以开发出更好的植物酶，这涉及到将酶插入微生物中，并将微生物的生长与酶的功能改进结合起来。具有更好酶的微生物生长更快，从而能够发现新的酶变种，例如，抗除草剂或可以促进有价值的化学物质的积累。CRISPR-Cas9允许在没有持久转基因的情况下编辑植物基因组。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。该项目是将通过持续定向进化开发更好的酶功能与CRISPR-Cas9相结合的原则证明。这两项技术将给农业带来革命性的变化。培训目标包括开发合成生物学方面的专门知识以补充现有技能，积极参与面向公众的科学宣传，以及完善沟通技能。更广泛的影响包括在佛罗里达大学帮助组织植物魅力日，以及志愿参加每一所佛罗里达学校的科学家计划，向学生介绍他们社区正在进行的研究。人类将植物用于食物、纤维、燃料、药品、工业化学品和其他目的。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的潜力仍然巨大。然而，在这项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，现在正在开辟植物代谢工程的新领域。可以使用连续的定向进化来快速探索酶适应性的前景，以连续合并多个有益的突变。CRISPR-Cas9使基因组编辑发生了革命性的变化，并导致了像TargetAID这样强大的新技术，它可以精确地编辑特定基因中的碱基对。该项目将利用CRISPR-CAS9在拟南芥中的持续定向进化和无缝编辑的力量，采用原则证明组合的方法。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将这些抗性突变导入拟南芥，而不使用持久的转基因盒。然后，这种结合的方法将被应用于将反馈不敏感的基因工程转化为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，结果将在任何可能的时候发表在开放获取的期刊上。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.