Maize Genome Modification: Technology and Applications

RNA guided CRISPR-Cas9 has rapidly become the technology of choice for maize genome editing due to its simplicity, efficiency and versatility. We have made several improvements to the commonly used SpCas9 genome editing system, including (1) the discovery and characterization of novel nucleotide-directed endonucleases with unique biochemical and biophysical properties, and (2) the development of marker-free, DNA-free, genome editing methodologies. We have established a robust genome editing process for maize that includes best practices for guide selection, Agrobacterium and biolistic optimized delivery of Cas9, Illumina sequencing approaches for examining genome editing activities, and transformation technologies that enable germplasm independent transformation. Using our pipeline, we have created non-transgenic hybrids with improved output characteristics and agronomic traits directly in elite germplasm. Additionally, we utilize this powerful tool to enable transgenic trait stacking and provide insight into gene expression by position effects.