Team:BOKU-Vienna/Description

Theory

V

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 evolution1,2. 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 mutations7,8.

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). 


Figure 1: Scheme of the design used by Crook et al. in 11.


Although having delivered impressive results such as a mutation rate of 0.15 kb-1­ the concept still holds room for improvement as argued by Zheng et al. in their review of targeted mutagenesis13.  For example, Ty1 as a mobile genetic element can reintegrate anywhere in the genome, preferred upstream of genes transcribed by RNA polymerase III14 possibly reducing selection efficiency due to the presence of multiple copies of the heterologous gene in a single cell. Additionally, in this setting the reverse transcriptase likewise is continuously mutated increasing the risk of inactivation as time carries on.

Drawing inspiration from this retroelement-based approach and having its potential weaknesses in mind we started thinking about the benefits of a more universal hypermutation strategy relying on reverse transcription of the gene of interest and reintegration of the generated cDNA such as:

  1. Site-specific reintegration would make sure to replace the original gene variant maintaining single-copy status for optimal selection behavior.
  2. Expression of the required reverse transcriptase in trans rather than within the synthetic retroelement eliminates the risk of inactivating the enzyme by mutation.

In such a system only the GOI would be mutated and remain being present in just a single copy. The overall scheme of our D.I.V.E.R.T. (directed in vivo evolution via reverse transcription) concept is depicted in Figure 2.


Figure 2: General scheme of the D.I.V.E.R.T. cycle


Theoretical considerations

To sum up: we wanted to build a fully synthetic retrotransposon-like genetic element that would allow our gene of interest to continuously undergo the retrotransposon life cycle accumulating mutations over time. The main events that need to be functionally implemented for such a system to work would be the in vivo reverse transcription carried out by a heterologous reverse transcriptase as well as site-specific recombination. For both processes several options are available. The reasoning that goes into our choices as well as some other thoughts are briefly explained below.