Many other strategies exist, such as removal of constitutive exons through NHEJ-mediated deletions with pairs of guide RNAs flanking these exons (Liu et al. 2016a, b), or using a homology construct that guides precise exon deletion of one allele (Fig. 4a), coupled with indel mutations on the other. These systems can also be used to make larger deletions to recapitulate for instance copy number variants (CNVs), which could be informative in certain cases. In all cases, it is also important to consider whether the knockout of the gene will be lethal at a cellular level, in which case conditional knockout by for instance flanking constitutive exons with recombinase sites, or conditional by inversion (COIN) or FLIP strategies (Economides et al. 2013; Andersson-Rolf et al. 2017) may be beneficial (Fig. 4c). Inducible CRISPR systems can also be employed to restrict mutations to particular points in time (Bertero et al. 2016), and may be helpful in certain situations.
