A new age for Synthetic Biology: L-SCRaMbLE, or, how to shuffle genome with light
Project SC2.0 is the first attempt of Synthetic Biology to produce a yeast strain with an entirely redesigned synthetic genome, still mantaining wild type phenotype. SCRaMbLE (Synthetic Chromosome Recombination and Modification by LoxP Mediated Evolution) is a recombination system implemented in the project to perform in vitro inducible and guided evolution. The development of a finely controlled Cre recombinase would ensure the tuning of SCRaMbLE dynamics or guarantees the isogenicity.
Synthetic genome is supposed to be minimal and stable, whereby it will be depleted of all hotspot of instability, such as trasposones, tRNA genes, tandem and subtelomeric sequences. In order to shorten the genome, researchers deleted all dispensable sequences, such as genes in high copy number, repetitive sequences, and homopolymer in genome. To generate genome flexibility and allow fine-tuned recombination thruogh SCRaMbLE system, 4000 LoxPsym sites were introduced all over the genome[1]. Sc2.0 implements LoxPsym sites, these sites differ from the original Batteriophage P1 LoxP sites since they contain a symmetric spacer region within the 34 bp long Cre recombination site. Cre-mediated recombination between two loxPsym sites can than lead to deletions or inversions of DNA fragments, with theoretically equal probability, in contrast with native LoxP site, by which the event of recombination were necessarily decided a priori by researcher, not allowing a random evolution [2].
As we said, the researcher introduced loxPsym in the genome to allow CRE-dependent random recombination. This process has originally been performed in the Sc2.0 through a recombinase fused with the Estradiol binding domain (EST-Cre). In presence of estradiol, EST-Cre can move to the nucleus, where it mediates recombination. It is than reliably inducible, and it can be turned down only by diluition of estradiol. To overcome this limitation Lena Hochrein and collaborators, developed a new light-mediated system of induction and tunability of recombination, called L-SCRaMbLE[3].
The team designed a Light-Cre recombinase fusing Cre with a photoreceptor of the Arabidopsis Thalliana. The new L-cre consists in a normal cre, splitted in two halves, CreC and CreN. The first half is fused to the PIF3 protein, that can bind the photoreceptor PhyB, which is fused to the CreN half. Briefly, if the chromofore PCB is provided to the medium, it can bind the PhyB protein, regenerating the photoreceptor. The excitation with a 660 nm wavelength (red) allow the conformational change of PhyB-PCB from the Pf to the Pfr state, enabling the binding to the PIF3 protein and re-assebling at the same time the cre recombinase complex.
The team reported a very low basal activity of L-Cre in the absence of light and high recombination capacity, in term of outcoming variability, after red light exposure. They also obseved an high reliable turn off efficiency, achieved through far red light exposure and re-plating the cells in a PCB free medium.
In the study L-Cre and EST-Cre were tested on a high loxPsym density episomal plasmid designed with 4 genes encoding the beta-karotene pathway, each flanked by 2 loxPsym sites. The recombinase activity and the different pattern of recombination was tested respectively through a colorimetric and a sequencing assay.
In our opinion a colorimetric assay is not fully reliable to investigate the actual occured recombination. Indeed some recombination events do not lead to colour change, as demostrated in the study itself, moreover the number of plasmid per cell is not controllabe, leading to possible compensation events.
Light-CRE has yet a very low activity compared to the original EST-Cre and requires a long time of induction. The activity of Light-Cre was not tested on LoxPsym sites dispersed on a chromosome or in a genome. We than question the efficiency of Light-Cre applied on a whole genome, in terms of time required for satisfing results. Indeed the researcher themselves were able to test the activity of EST-Cre on a 2 loxP sites flanked gene located on a chromosome, but consider unreliable testing L-Cre on the same target, in reason of the about 20 time lower activity of L-Cre comparing to EST-Cre, reported in previous experiment. SCRaMbLE system in Sc2.0 project requires genome-scale recombination events to occur,Itis possible that Light-Cre, applied to restriction sites dispersed on genome, would shown a too low efficiency.
The confrontation of L-cre and EST-cre are based on data obtained from a 10 second light induction every 5 minutes for L-cre, while EST-Cre was obviously always stimulated by continuous of estradiol. The researcher didn’t actually justify the choice of the protocol of stimulation, so the difference of the levels of recombination between the two stimulated enzymes could be caused by a different magnitude of response of the two different enzymes for respective induction protocol. If we consider that 1 µM of estradiol could activate EST-cre much more that how 25 µM of PCB can activate L-cre, because of different costant of affinity of the different proteins, that would cause a higher activation of EST-cre, making It act more frequently on the DNA. So even if the recombination events have the same propability to happen, if the enzyme is more active, more events take place, making deletion more likely to happen, justifying why there’s such a gap between white colonies level between light and est cre. This aspect would not justify the gap of background activity.
In short, according to us, an investigationt of L-cre on the target It was meant for (the yeast syntethic whole genome) is required, in order to define L-SCRaMbLE as a reliabile tool for controlled evolution in Sc2.0.
References
- Richardson, Sarah M., et al. “Design of a synthetic yeast genome.” Science 355.6329 (2017): 1040-1044.
- Pretorius, I. S., and J. D. Boeke. “Yeast 2.0—connecting the dots in the construction of the world’s first functional synthetic eukaryotic genome.” FEMS yeast research 18.4 (2018): foy032.
- Hochrein, Lena, et al. “L-SCRaMbLE as a tool for light-controlled Cre-mediated recombination in yeast.” Nature communications 9.1 (2018): 1931.
- http://syntheticyeast.org/sc2-0/related-technology/evolution-experiments/