Skip to content

When you choose to publish with PLOS, your research makes an impact. Make your work accessible to all, without restrictions, and accelerate scientific discovery with options like preprints and published peer review that make your work more Open.

PLOS BLOGS The Official PLOS Blog

I am known by many names, but it’s all SCRAMBLEd


Guest post by Thomas Clements


SCRAMBLE is a new form of directed evolution pioneered in Sc 2.0, an entirely synthetic form of Saccharomyces cerevisiae, to rapidly engineer the genome. Recently, 8 new publications in Nature Communications report improvements to this method and many have a unique anagram to show for it.



Slow, incremental changes are a hallmark of evolution, which is why the development of new species, or speciation, is such a slow process. However, smaller changes on the DNA level happen much more quickly. Thousands of these small mutations accumulate over a very long period of time until a new species is created. In directed evolution, evolution is sped up toward a specific end goal through multiple rounds of mutagenesis, selecting desired traits, and then amplifying these desired traits. Scientists are working to accelerate this process even further in the baker’s yeast Saccharomyces cerevisiae in a form of directed evolution known as Synthetic Chromosome Rearrangement And Modification BLoxP-mediated Evolution or SCRAMBLE.

This work is largely possible through the Synthetic Yeast Genome Project (Sc 2.0), whose goal is to create the first eukaryotic synthetic genome. Sc 2.0 differs from wild-type S. cerevisiae in many ways, but perhaps the most significant is that a 34 base pair (bp) loxP site is placed every 10,000 base pairs in the 3’ untranslated region (UTR), which is non-essential, in every gene. This site lets scientists specifically express the Cre recombinase to recombine these loxP sites to result in mutations in the DNA in form of deletions, insertions, translocations and inversions at distinct locations in the yeast genome, as shown in figure 1. SCRAMBLE simply is the use of the CRE recombinase in the Sc 2.0 yeast (Dymond et al., 2011).

Diagram of the SCRAMBLE method in synthetic yeast chromosome III showing an example of an inversion, translocation, and a deletion as a result of Cre recombinase activity (Annaluru et al., 2015) CC-BY 4.0.

Read more: Extreme makeover yeast edition: de novo synthesis of five chromosomes

Recently, there have been 8 new publications about the SCRAMBLE technique in Nature Communications and we will go over each of them by category: improvements to the system or a new tool to accentuate the method.


Improvements to SCRAMBLE

 Use of diploid strains

One of the disadvantages of the initial SCRAMBLE method was the high mortality rate. This was due to the fact that haploid—with a single set of chromosomes—cells that had their genomes rearranged due to SCRAMBLE did not have a second set of chromosomes to compensate for this loss. Thus, mutations in necessary genes caused the cells to perish. In order to minimize this effect, scientists used strains of yeast that are heterozygous diploids, which have one wild-type set of chromosomes and one set of chromosomes with at least one synthetic chromosome, in order to increase survivability (Shen et al., 2018).

However, haploid cells are still suitable for SCRAMBLEing. Scientists used (haploid) S. cerevisiae strains that had a completely synthetic chromosome V (Blount et al., 2018) to improve violacein and penicillin biosynthesis, as well as xylose utilisation. Furthermore, they established a new method to map the rearrangements using long-read nanopore sequencing.

Method Improvements

Advancing the system, scientists have shown a way to externally optimize a specific pathway while simultaneously engineering the genome in a new method known as SCRAMBLE-in (Liu et al., 2018). Here, the SCRAMBLE is done in vitro (in test tubes) and then the genetically altered strains are transferred into wild-type S. cerevisiae strains. This is advantageous to the previous iteration of SCRAMBLE because the process of SCRAMBLE-in is highly controllable in vitro and the subsequent analysis is more straightforward. A very similar method of using in vitro SCRAMBLE was shown to optimize yield of the β-carotene pathway (Wu et al., 2018).

Next, scientists began focusing on the yield of the pathways they are optimizing and developed a new technique known as Multiplexed SCRAMBLE Iterative Cycling (MuSIC) (Jia et al., 2018). Here, host strains are subjected 5 iterative rounds of SCRAMBLE to enhance product yield of specific pathways. Haploid strains were reported to have a 1.5 yield increase of product yield, while diploid strains had up to a 30-fold yield increase.


Schematic overview of two L-SCRaMbLE systems. Both systems rely on the dimerization of the two Cre nuclease fragments. a  In darkness or far-red light (λ = 740 nm), CreC (C-terminal) is located in the nucleus, while CreN  (N-terminal) is located in the cytoplasm. Upon red light illumination (λ = 660 nm) CreN binds CreC in the cytoplasm (not shown), thereby reconstituting Cre recombinase from its split halves. Reconstituted Cre moves to the nucleus where it induces recombination between the loxPsym sites. b  In darkness or far-red light (λ = 740 nm) CreC and CreN are separately located in the nucleus. Upon red light illumination (λ = 660 nm) CreN binds CreC, thereby reconstituting Cre recombinase from its split parts, allowing recombination at loxPsym sites. Image from Hochrein et al. 2018 (CC-BY 4.0)

Finally, two additional manuscripts report new tools to be used with the SCRAMBLE system. The first is a tool to rapidly select and characterizing yeast that have been SCRAMBLED known as Reporter of SCRAMBLEd Cells using Efficient Selection (ReSCuES) (Luo et al., 2018). Here, scientists identify SCRAMBLEd populations by incorporating two selective markers, the first is expressed before SCRAMBLE and the second is only active after the activity of the Cre recombinase. The second tool, known as L-SCRAMBLE, uses a light controlled Cre (as opposed to the typical estradiol induced) to result in a more finely controlled recombination (Hochrein et al., 2018). Here, scientists report a 179-fold induction upon exposure to red light, thus indicating the increased control of using L-SCRAMBLE.



Overall, the SCRAMBLE method has broad implications in synthetic biology, metabolic engineering and genome engineering. Possible concerns about the technology are countered by possible trade-offs such as slow growth and stability of these engineered strains. In the future, novel improvements to SCRAMBLE could lead to discovering interactions that rely on multiple genes being mutated simultaneously, which can then expand to exciting new technologies. Nonetheless, this is just the beginning, according to Jef Boeke, director of the Institute for Systems Genetics at New York University’s Langone Health, who is in charge of a large international team devoted to creating all 16 synthetic yeast chromosomes by 2020. They are well on their way, with currently 6 of the 16 chromosomes in S. cerevisiae synthesized. By undertaking this project and with all of the improvements to SCRAMBLE, scientists will be able to engineer the entire yeast genome quicker than ever before and lead to numerous applications including optimizing the natural synthesis of specific products and also applying these lessons in engineering other organisms.



Annaluru, N., Ramalingam, S., and Chandrasegaran, S. (2015). Rewriting the blueprint of life by synthetic genomics and genome engineering. Genome Biol. 16, 125.

Blount, B.A., Gowers, G.-O.F., Ho, J.C.H., Ledesma-Amaro, R., Jovicevic, D., McKiernan, R.M., Xie, Z.X., Li, B.Z., Yuan, Y.J., and Ellis, T. (2018). Rapid host strain improvement by in vivo rearrangement of a synthetic yeast chromosome. Nat. Commun. 9, 1932.

Dymond, J.S., Richardson, S.M., Coombes, C.E., Babatz, T., Müller, H., Annaluru, N., Blake, W.J., Schwerzmann, J.W., Dai, J., Lindstrom, D.L., et al. (2011). Synthetic chromosome arms function in yeast and generate phenotypic diversity by design. Nature 477, 471–476.

Hochrein, L., Mitchell, L.A., Schulz, K., Messerschmidt, K., and Mueller-Roeber, B. (2018). L-SCRaMbLE as a tool for light-controlled Cre-mediated recombination in yeast. Nat. Commun. 9, 1931.

Jia, B., Wu, Y., Li, B.-Z., Mitchell, L.A., Liu, H., Pan, S., Wang, J., Zhang, H.-R., Jia, N., Li, B., et al. (2018). Precise control of SCRaMbLE in synthetic haploid and diploid yeast. Nat. Commun. 9, 1933.

Liu, W., Luo, Z., Wang, Y., Pham, N.T., Tuck, L., Pérez-Pi, I., Liu, L., Shen, Y., French, C., Auer, M., et al. (2018). Rapid pathway prototyping and engineering using in vitro and in vivo synthetic genome SCRaMbLE-in methods. Nat. Commun. 9, 1936.

Luo, Z., Wang, L., Wang, Y., Zhang, W., Guo, Y., Shen, Y., Jiang, L., Wu, Q., Zhang, C., Cai, Y., and Dai, J. (2018) Identifying and characterizing SCRaMbLEd synthetic yeast using ReSCuES. Nat. Commun. 9, 1930.

Shen, M.J., Wu, Y., Yang, K., Li, Y., Xu, H., Zhang, H., Li, B.-Z., Li, X., Xiao, W.-H., Zhou, X., et al. (2018). Heterozygous diploid and interspecies SCRaMbLEing. Nat. Commun. 9, 1934.

Wu, Y., Zhu, R.-Y., Mitchell, L.A., Ma, L., Liu, R., Zhao, M., Jia, B., Xu, H., Li, Y.-X., Yang, Z.-M., et al. (2018). In vitro DNA SCRaMbLE. Nat. Commun. 9, 1935.

Banner Image: Yeast. Image from Flickr CC-BY 2.0


About the Author:

Thomas Clements received his Ph.D. at Rice University in 2018 and will be transitioning to a career as a Lecturer in the Biological Sciences Department at Vanderbilt University starting in Fall 2018. He is also a member of the Early Career Scientist Policy and the Education Committee at the Genetics Society of America. His research centers on improving CRISPR-Cas9 gene editing in zebrafish, but he is also passionate about science literacy and inspiring the next generation of scientists to pursue careers that align with their passions.


Back to top