Since the discovery of the CRISPR/Cas system and its in vivo application for site-specific targeted mutagenesis, this technique is wildly used in a great variety of organisms, such as many plant species. Commonly used for this application is the Cas9 enzyme from Streptococcus pyogenes. Here, we describe the application of two Cas9 orthologs from Streptococcus thermophilus and Staphylococcus aureus for targeted non-homologous end-joining mediated mutagenesis in Arabidopsis thaliana. With both orthologs, we could show efficient inheritance of the induced mutations. As both Cas9 orthologs are smaller in size than the enzyme of S. pyogenes and as the Protospacer adjacent motifs (PAMs) differ between all orthologs, they are attractive alternative tools for genome engineering in plants.
Keywords: CRISPR/Cas; Double-strand break repair; Gene editing; Genome engineering; Targeted mutagenesis.