Another important consideration is to minimise off-target mutagenesis, the extent of which is still debatable in the field, and likely depends on the exact system used to introduce the CRISPR reagents (Cradick et al. 2013; Fu et al. 2013; Veres et al. 2014). What is clear is that some mismatches between the guide RNA and target DNA can be tolerated, and the degree to which they impact on endonuclease activity depends on their position within the sequence, with those nucleotides closer to the PAM sequence playing a more critical role in target recognition (Hsu et al. 2013). Careful design of crRNA target sites to avoid off-targets of less than 3 mismatches can be readily achieved using a variety of online tools and will certainly minimise any potential problems. Methods for improving specificity have been developed using either pairs of Cas9 enzymes each of which is unable to generate a DSB alone (Guilinger et al. 2014; Ran et al. 2013; Tsai et al. 2014), truncated guide RNAs (Fu et al. 2014) or protein engineering of Cas9 to improve specificity (Kleinstiver et
