Fig. 1: Evolution of PE variants with enhanced activity in OrthoRep.

a Parallel evolution of prime editors (PE) by culturing yeast cells in 96 well plates over four subsequent rounds in L-histidine-depleted (-L-His) selection media. In the first round of evolution, outgrowing yeast cells were normalized prior to extraction of the linear plasmid (p1) via PCR and transformation into fresh host cells for the second round. The same procedure was repeated for the third and fourth round. b PE variants containing mutations that increase prime editing rates are enriched in selective conditions: a stop codon in front of the auxotrophic marker Imidazoleglycerol-phosphate dehydratase (HIS3) must be repaired by prime editing on the nuclear plasmid co-expressing the prime editing guide RNA (pegRNA) for successful yeast growth in selective conditions. c Effect of evolved PE variants on yeast growth quantified as optical density at 600 nm (OD600) under selective conditions. The assessed variants contained the following mutations: PE_Y17 (nCas9 S219A, RT K445T), D2 (nCas9 A259D), A10 (RT Y64W, K373R, R389C, L432M, K445T), E11 (RT Y64W), B4 (RT R44H), H8 (nCas9 S219A, RT K445T), G3 (nCas9 A259D, linker G30S, RT R389C, S606A), H1 (nCas9 A259D, RT Y64W), H3 (nCas9 A259D) E5 (nCas9 S219A, RT K445T), A1 (nCas9 A259D, linker G30S, RT R389C, S606A), A5 (nCas9 S219A, RT Y64C, K445T), A8 (nCas9 S219A, linker G30S, RT K445T), F6 (nCas9 A259D, RT R44H), E3 (nCas9 S320R), B3 (nCas9 R71C, A259D, linker G30S, RT K445T), A6 (nCas9 S318N, RT R389C, L432M), B8 (nCas9 S320N,RT K373R), C8 (nCas9 Y132C), G5 (nCas9 A259D,RT K373R), A2 (nCas9 S219A, RT K373R, K445T). Data are displayed as the mean of two individual replicates. d Schematic of the in vitro assay to assess PE kinetics (PEKIN). PE variants are subcloned as self-cleaving peptide fused to a green fluorescent protein (P2A-GFP) and transfected into HEK293T cells. Cells are lysed and PE expression levels are normalized by fluorescence intensity. An in vitro transcribed (IVT) pegRNA forms the RNP with the PE, which is incubated with a synthetic dsDNA substrate. e Schematics illustrating the strategy used to quantify PE activity in PEKIN via quantitative PCR (qPCR). f Quantified prime editing activities of the 21 PE variants isolated after four rounds of evolutions in OrthoRep relative to PEmax. Product formation of the variants is relative to the product formed by PEmax and was performed as a single replicate (n = 1).