SCRaMbLE generates designed combinatorial stochastic diversity in synthetic chromosomes
- Yue Shen1,
- Giovanni Stracquadanio2,
- Yun Wang1,
- Kun Yang2,
- Leslie A Mitchell3,
- Yaxin Xue1,
- Yizhi Cai4,
- Tai Chen1,
- Jessica S Dymond2,
- Kang Kang1,
- Jianhui Gong1,
- Xiaofan Zeng1,
- Yongfen Zhang1,
- Yingrui Li1,
- Qiang Feng1,
- Xun Xu1,
- Jun Wang1,
- Jian Wang1,
- Huanming Yang1,
- Jef D Boeke3 and
- Joel S Bader2,5
- ↵* Corresponding author; email: joel.bader{at}jhu.edu
Abstract
Synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) generates combinatorial genomic diversity through rearrangements at designed recombinase sites. We applied SCRaMbLE to yeast synthetic chromosome arm synIXR (43 recombinase sites), and then use a computational pipeline to infer or unscramble the sequence of recombinations that created the observed genomes. Deep sequencing of 64 synIXR SCRaMbLE strains revealed 156 deletions, 89 inversions, 94 duplications, and 55 additional complex rearrangements; several duplications are consistent with a double rolling circle mechanism. Every SCRaMbLE strain was unique, validating the capability of SCRaMbLE to explore a diverse space of genomes. Rearrangements occurred exclusively at designed loxPsym sites, with no significant evidence for ectopic rearrangements or mutations involving synthetic regions, the 99% non-synthetic nuclear genome, or the mitochondrial genome. Deletion frequencies identified genes required for viability or fast growth. Replacement of 3′ UTR by non-UTR sequence had surprisingly little effect on fitness. SCRaMbLE generates genome diversity in designated regions, reveals fitness constraints, and should scale to simultaneous evolution of multiple synthetic chromosomes.
- Received April 22, 2015.
- Accepted November 12, 2015.
- Published by Cold Spring Harbor Laboratory Press
This manuscript is Open Access.
This article, published in Genome Research, is available under a Creative Commons License (Attribution 4.0 International license), as described at http://creativecommons.org/licenses/by/4.0/.
