Directed Evolution – a (very) short overview

After decades of continuous methodological advancement and increasing understanding of biomolecule structure and functionality directed evolution nevertheless is still the most powerful method in protein or aptamer engineering. Classic in vitro strategies, however, require substantial effort in terms of lab work and time investment to perform several consecutive rounds of evolution^1,2. In order to automate the laborious process scientists have tried to devise systems that traverse the four steps of Darwinian evolution (mutation, expression, selection, replication) in a continuous cycle in vivo (reviewed in ³). In the earliest approaches this was achieved by simply cloning the gene of interest into mutator E. coli strains⁴ showing reduced DNA replication fidelity or into E. coli strains carrying inducible mutator plasmids⁵. While these settings proved to be useful for the generation of complex multifactorial phenotypes like organic solvent tolerance⁵ they cannot provide the regional selectivity that is desired in single-gene protein evolution as globally enhanced mutagenesis leads to slow growth and reduced transformation efficiency⁶ in addition to obscured phenotypic expression due to unwanted off-target mutations^7,8.

Therefore, the ideal system for in vivo directed evolution would avoid those side effects by subjecting the host organism to locally confined hypermutation. Such an arrangement would allow the researcher to rapidly mutate and evolve a defined single sequence or gene while leaving the rest of the genome unchanged. Several strategies to locally constrain enhanced mutation rates have been devised so far; including plasmids harboring regions with low replication fidelity⁹, elaborate phage-assisted systems confining the accumulation of mutations to the phage genome while keeping the overall mutational load in the cell population in a steady state¹⁰ or retrotransposon-based methods¹¹. Although definitely representing a large step towards the right direction, still none of the designs mentioned allow the continuous mutation of a single copy of a single gene in vivo. With D.I.V.E.R.T. we want to build a system that does.

The D.I.V.E.R.T concept

In their work published in 2016 Crook et al. probably generated the very first retrotransposon-based system for in vivo directed evolution by inserting a gene of interest into a truncated version of the native Ty1 retrotransposon in S. cerevisiae (Figure 1)¹¹. Thus, the GOI is subjected to the retrotransposon life cycle and continuously mutated due to the error-prone nature of Ty1 reverse transcriptase (low fidelity is inherent to most reverse transcriptases¹²).