Targeted genome modifications are important for both fundamental and applied research. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR-associated protein 9) technology has been successfully used in various plant species with high efficiency. Approaches with paired Cas9 nickase enhance the specificity of the CRISPR/Cas9 system by using guide RNA pairs to create two staggered single strand breaks on complementary DNA strands. Here we used maize mesophyll protoplasts as a transient test system and demon- strated the mutagenic potential of Cas9 nickases. Although we found activity for all the three different guide RNA pairs tested, their efficiency varied considerably. Characterization of the modification events revealed a high ratio of large deletions as well as insertions of donor DNA fragments. By the use of the maternally expressed in embryo
1 gene (mee1) as model target sequence, we could demonstrate that transcriptionally inactive and methylated genomic loci are practical targets of Cas9 nickase. The high specificity of Cas9 nickase approaches might provide advantage for genome modifications of certain loci in the complex and highly repetitive maize genome
