Targeted Gene Editing in Tetraploid Switchgrass (Panicum virgatum L.) by CRISPR/Cas9

The CRISPR/Cas9 system is a simple yet powerful tool to edit plant genomes in a targeted manner. Switchgrass (Panicum virgatum L.) is a self-incompatible, perennial grass species that has been designated a model bioenergy crop by the U.S. Department of Energy due to its high yielding potential. To enhance breeding efforts of switchgrass we explored the feasibility of using CRISPR/Cas9 in the tetraploid cultivar ‘Alamo’ in switchgrass through both transient assay and stable transformation. For transient assay, a non-functional Green Fluorescent protein (GFP) gene was successfully mutated by a Cas9/sgRNA complex and its function restored. Stable transformation with CRISPR/Cas9 construct targeting Flowering Locus T (FT), APETALA 1 (AP1), Brassinosteroid Receptor Insensitive 1 (BRI1) and Phosphoglycerate Mutase (PGAM) genes were carried out. With the pEnter4:gRNA-PGAM/Cas9 construct, targeting the PGAM gene, a mutation frequency of 13.7% was obtained. Among the first generation of mutated plants (T₀), six are heterozygous, and one is homozygous for a 1bp deletion caused by CRISPR/Cas9. Our results demonstrate for the first time that CRISPR/Cas9 system can be successfully used in switchgrass genome editing.