Fig. 2

In vivo continuous evolution strategies. a OrthoRep for error-prone and orthogonal DNA replication; The error-prone DNA polymerase (DNAP) (p1pol) does not interact with the host genome, instead mutating only genes of interest during their replication. DNA polymerase (p2pol) also replicates a needed accessory plasmid. b In vivo continuous evolution (ICE) method for reverse-transcription based mutagenesis in three steps; (1) Genetic cargo and other elements are flanked by long terminal repeats (LTR) of a Ty1 retrotransposon and inserted into the genome. (2) After its transcription, the cargo is reverse transcribed by an error-prone reverse transcriptase into cDNA, introducing mutations. (3) The mutated cargo cDNA is integrated into the genome. c Eukaryotic multiplex automated genome engineering in yeast (eMAGE); libraries of ssODNS, which bind and introduce mutations into the lagging strand of DNA in the replication fork, are inserted into yeast cells, such that genes, promoters, etc. can be edited in yeast cells over multiple cycles, leading to a wide range of genetic variation. For these in vivo continuous evolution strategies, variants with mutated genotypes are screened for improved phenotypes, and if necessary, mutated further over more evolutionary rounds