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 al. 2016; Slaymaker et al. 2016). Some of these strategies including the double nickase approach (Ran et al. 2013) have been successfully used in hiPS cells (Eggenschwiler et al. 2016; Wu et al. 2016b), although these systems will never remove the potential for off-target mutations completely. Therefore at least in the case of disease models, where a small number of lines are produced, it is also possible to sequence the putative off-target sites (or even the whole genome), generate cell lines with independent guides each of which would have a different set of off-targets, or perform another round of genome engineering to repair the introduced genetic change.
