Supplementary Figure 3: Detailed synonymous codon spreading strategy to enable mutation of codons outside of guide RNA recognition regions

Amino acid saturation editing strategy for open reading frames with (a) target codons that fall within the 20 bp of NGG-PAM guide recognition sequences and (b) target codons that fall outside of the nearest NGG-PAM guide recognition sequence. The examples show the design of the donor DNA to accompany guide RNA utilizing the nearest downstream PAM. The nonsynonymous changes (red) are accompanied by synonymous changes (cyan) that spread towards the Cas9 cleavage site (3 bp upstream of the PAM). The synonymous changes block cleavage of the donor by the Cas9::guide complex and minimize microhomology between the nonsynonymous variant of interest and the guide-disrupting changes. A pseudo-WT control is included to rule out effects due to the synonymous changes.