Efficient genome editing and gene knockout in <i>Setaria viridis</i> with CRISPR/Cas9 directed gene editing by the non-homologous end-joining pathway

Abstract

The CRISPR/Cas9 system has been used for genome editing in several organisms, including higher plants. This system induces site-specific mutations in the genome based on the nucleotide sequence of engineered guide RNAs. The complex genomes of C4 grasses makes genome editing a challenge in key grass crops like maize (<i>Zea mays</i>), sorghum (<i>Sorghum bicolor</i>), <i>Brachiaria</i> spp., switchgrass (<i>Panicum virgatum</i>), and sugarcane (<i>Saccharum</i> spp.). <i>Setaria viridis</i> is a diploid C4 grass widely used as a model for these C4 crop plants. Here, an optimized CRISPR/Cas9 binary vector that exploits the non-homologous end joining (NHEJ) system was used to knockout a <i>green fluorescent protein</i> (<i>gfp</i>) transgene in <i>S. viridis</i> accession A10.1. Transformation of embryogenic callus by <i>A. tumefaciens</i> generated ten glufosinate-ammonium resistant transgenic events. In the T0 generation, 60% of the events were biallelic mutants in the <i>gfp</i> transgene with no detectable accumulation of GFP protein and without insertions or deletions in predicted off-target sites. The <i>gfp</i> mutations generated by CRISPR/Cas9 were stable and displayed Mendelian segregation in the T1 generation. Altogether, the system described here is a highly efficient genome editing system for <i>S. viridis</i>, an important model plant for functional genomics studies in C4 grasses. Also, this system is a potential tool for improvement of agronomic traits in C4 crop plants with complex genomes.