Fig. 3

CRISPR-associated substrate-linked directed evolution (CaSLiDE). a Schematic illustration of CaSLiDE for base editing. The process starts by cloning a base editor enzyme library (blue) into the pEVO expression vector, which contains expression cassettes for sgRNAs (gray) and sgRNA target sites (yellow). After expression of the base editors, plasmids are isolated and digested with restriction enzymes, which target the sgRNAs and the sgRNA target sites. Applying a restriction digest to the plasmids results in one linear fragment for the edited plasmids and in two fragments for non-edited plasmids. An error-prone PCR using indicated primers (arrows) exclusively generates products of the edited plasmids. These amplified Un1Cas12f1-ABE variants are then subjected to the next evolution cycle. b Schematic illustration of the base editing plasmid assay. c Plasmid-based assays of the WT Un1Cas12f1-ABE at indicated L-arabinose induction levels. Target sites 1–3 were digested separately by using the corresponding restriction enzymes NdeI, HpaI, or PsiI, respectively. d Plasmid-based assay of the evolution starting point and the final evolved library, triple digested simultaneously with NdeI, HpaI, and PsiI. Note the presence of a slower migrating band for the evolved library at indicated L-arabinose induction levels, representing fully edited plasmids