It is important that frameshifts are not made too early in the protein coding sequence, since if the open reading frame (ORF) is too short, reinitiation at a downstream start codon can occur (Zimmer et al. 1994). The efficiency of such reinitiation is dependent on the length of the upstream ORF, and is highly inefficient if this is greater than 30–40 amino acids (Luukkonen et al. 1995). Equally, frameshifts should not be introduced too late in the coding sequence. Normally premature stop codons are recognised by the cell and trigger the process of nonsense mediated decay (NMD), which prevents expression of the entire protein through mRNA degradation (Hug et al. 2016). However, if the premature stop codon is present in the final exon, this process does not occur, and a slightly truncated peptide will be produced, likely with at least partial functionality (Hug et al. 2016). In order to obtain a complete knockout allele, it is therefore optimal to target a constitutive exon at least 30–40 amino acids into the protein coding sequence that is not in the final exon.
