EAGER: Heritable Gene Editing in Maize using Transient Expression of the CRISPR-Cas9 Endonuclease System

University Of Florida, Gainesville FL

Investigators

Abstract

The growth and productivity of crop plants are fundamentally determined by DNA. Throughout the history of agriculture, farmers and breeders have selected for variants in DNA sequence that improve plant productivity. Recent technologies, collectively known as gene editing, can induce DNA variants at specific sites in the genome. Gene editing can produce desired traits in crops, but the current process for gene editing in corn is technically challenging with limited throughput. This project will explore methods to more efficiently induce DNA variants at specific sites in the corn genome. The research will test whether short-term delivery of gene editing enzymes to corn stem cells will cause DNA variants that can be inherited from the treated plant to the next generation. If successful, the project will develop a new tool for genome research and corn breeding that is expected to have a high impact on understanding gene function and developing more productive corn varieties. The project will also directly involve undergraduates in the research to support the education and professional development of the next generation of scientists. The long-term goal of this research is to develop more efficient methods to edit the maize genome. Gene editing using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system is very effective at generating heritable mutations when the CRISPR system is introduced as a stable transgenic locus. This project will investigate whether transient expression of the Cas9 endonuclease and a synthetic, single guide RNA (gRNA) in shoot apical meristem cells can produce inheritable gene edits. The central hypothesis is that transient expression of Cas9 and gRNA in plant stem cells will induce mutant sectors large enough to contribute to the gametes. If successful, transient mutagenesis will produce targeted mutations without tissue culture or the generation of a transgenic organism. Aim 1 will determine if Agrobacterium tumefaciens infiltration of seedling shoots can transiently express foreign DNA in the shoot apical meristem at sufficient levels to cause targeted mutations with the CRISPR-Cas9 system. Aim 2 will determine if cell penetrating peptides can transfer DNA, protein, and gRNA into shoot stem cells at sufficient levels to cause targeted mutations.