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 the latter case, there are sequence requirements on where the piggyBac sequences can be integrated that may limit the effectiveness of this strategy (Yusa 2013). A third strategy involves a two-step genome editing strategy whereby in the first step the desired mutation is introduced alongside secondary mutations to prevent recutting, and subsequently, the secondary mutations are removed by a redesigned guide (or alternative Cas9 enzyme) and homology template (Fig. 4f) (Paquet et al. 2016; Kwart et al. 2017). However, these latter strategies involve considerably more complex processes, and at least two stages of clonal selection, increasing the time and cost of producing each mutation.
