SCRaMbLE generates designed combinatorial stochastic diversity in synthetic chromosomes
- Yue Shen1,2,9,
- Giovanni Stracquadanio3,4,9,
- Yun Wang1,9,
- Kun Yang3,
- Leslie A. Mitchell3,5,
- Yaxin Xue1,
- Yizhi Cai2,
- Tai Chen1,
- Jessica S. Dymond3,
- Kang Kang1,
- Jianhui Gong1,
- Xiaofan Zeng1,
- Yongfen Zhang1,
- Yingrui Li1,
- Qiang Feng1,
- Xun Xu1,
- Jun Wang1,6,7,
- Jian Wang1,
- Huanming Yang1,8,
- Jef D. Boeke5 and
- Joel S. Bader3,4
- 1BGI-Shenzhen, Shenzhen 518083, China;
- 2Centre for Synthetic and Systems Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3JL, United Kingdom;
- 3High-Throughput Biology Center, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, USA;
- 4Department of Biomedical Engineering, School of Engineering, Johns Hopkins University, Baltimore, Maryland 21218, USA;
- 5Department of Biochemistry and Molecular Pharmacology and Institute for Systems Genetics, NYU Langone Medical Center, New York, New York 10016, USA;
- 6Department of Biology, University of Copenhagen, DK-2200 Copenhagen, Denmark;
- 7Princess Al Jawhara Center of Excellence in the Research of Hereditary Disorders, King Abdulaziz University, Jeddah 21589, Saudi Arabia;
- 8James D. Watson Institute of Genome Science, Hangzhou 310058, China
- Corresponding authors: joel.bader{at}jhu.edu, jef.boeke{at}nyumc.org, yanghuanming{at}genomics.cn
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↵9 These authors contributed equally to this work.
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 used 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% nonsynthetic 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.
Footnotes
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[Supplemental material is available for this article.]
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Article published online before print. Article, supplemental material, and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.193433.115.
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Freely available online through the Genome Research Open Access option.
- Received April 22, 2015.
- Accepted November 12, 2015.
This article, published in Genome Research, is available under a Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0/.
