Targeted mutagenesis in the tetraploid switchgrass cultivar 'Alamo' using CRISPR-Cas9
Switchgrass (Panicum virgatum L.) is a highly self-incompatible perennial grass species which has been designated as a model bioenergy crop by the U.S. Department of Energy. To enhance breeding efforts of switchgrass we tested the efficacy of the targeted gene editing tool CRISPR-Cas9 in the tetraploid cultivar ‘Alamo’. Agrobacterium mediated transformation of embryogenic callus tissue averaged a transformation efficiency of 12.0% across experiments targeting four genes: Flowering Locus T (FT), APETALA 1 (AP1), Brassinosteroid Receptor Insensitive 1 (BRI1), and Phosphoglycerate Mutase (PGM). Out of 77 PCR confirmed transgenic events, a single event contained a heterozygous, CRISPR-Cas9 mediated 1 base pair deletion in the PGM target gene. Transgene silencing due to switchgrass’s highly methylated genome may play a role in the low mutation frequency. The mutation was obtained using a single sgRNA, however multiplex CRISPR using 2 sgRNA’s as well as tRNA constructs were also tested.