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

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.