Scarless mutagenesis is possible in some cases where the SNP happens to fall within the PAM sequence, or within a few nucleotides of the 3′ end of the guide RNA, since even single-point mutations at these sites can prevent re-cleavage. However, this is not the case in many instances, and certain nucleotides are therefore inaccessible to such manipulations at least with Streptococcus pyogenes Cas9. As mentioned above, orthologues from other species (Hou et al. 2013; Ran et al. 2015; Zetsche et al. 2015) or engineered variants (Kleinstiver et al. 2015a, b) are already increasing the range of potential targets, and it is likely that in the future, most SNPs will be amenable to manipulation in this manner. Another strategy is to use a larger dsDNA donor to introduce a selectable marker cassette into a non-functional region neighbouring the SNP of interest (Fig. 4e) (Wang et al. 2017; Yusa 2013). This cassette can subsequently be removed either by site-specific recombinases, or scarlessly by a second round of homologous recombination or the piggyBac transposase (Wang et al. 2017; Yusa 2013). However, in
