“Figure 5” (Figure to accompany Box 1).

Illustration of the probability calculations for non-silent bystander editing in a protein-coding gene, as described in Box 1. (a) Use of base editing to generate a non-silent mutation in the protein-coding region of a gene. There are three possible orientations of the base editor activity window relative to the reading frame of the protein. Base editors currently generate transition mutations, and the vast majority (97%) of transitions at codon position 3 are silent. Thus, the target base in a coding gene is predominantly located at coding position 1 or 2 (N¹ or N²), because the consequence of generating a transition at N³ is almost always to generate a silent mutation. In a 5-nt activity window, typical of a CBE, an average of 2.33 nucleotides at positions N¹ or N² are present in the window, in addition to the target nucleotide. For a 4-nt window typical of an ABE there are on average 1.75 nucleotides at codon positions 1 or 2 in addition to the target nucleotide (see Box 1). These additional editable nucleotides at N¹ or N², depending on their identity and sequence context, may contribute to bystander editing (see Box 1). (b) Flow chart to calculate the probability of finding a bystander mutation within a 5-nt window, as described in Box 1. The probability of the indicated event (p) assumes a genome with randomly distributed bases. For a description of how each probability was calculated, see Box 1. The same calculation is described for a 4-nt window typical of ABEs in Box 1.