title: SCRaMbLE generates designed combinatorial stochastic diversity in synthetic chromosomes.
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.
authors.affiliation: BGI-Shenzhen, Shenzhen 518083, China; Centre for Synthetic and Systems Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3JL, United Kingdom;
authors.forename: Yue
authors.initials: Y
authors.lastname: Shen
authors.affiliation: High-Throughput Biology Center, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, USA; Department of Biomedical Engineering, School of Engineering, Johns Hopkins University, Baltimore, Maryland 21218, USA;
authors.forename: Giovanni
authors.initials: G
authors.lastname: Stracquadanio
authors.affiliation: BGI-Shenzhen, Shenzhen 518083, China;
authors.forename: Yun
authors.initials: Y
authors.lastname: Wang
authors.affiliation: High-Throughput Biology Center, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, USA;
authors.forename: Kun
authors.initials: K
authors.lastname: Yang
authors.affiliation: High-Throughput Biology Center, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, USA; Department of Biochemistry and Molecular Pharmacology and Institute for Systems Genetics, NYU Langone Medical Center, New York, New York 10016, USA;
authors.forename: Leslie A
authors.initials: LA
authors.lastname: Mitchell
authors.affiliation: BGI-Shenzhen, Shenzhen 518083, China;
authors.forename: Yaxin
authors.initials: Y
authors.lastname: Xue
authors.affiliation: Centre for Synthetic and Systems Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3JL, United Kingdom;
authors.forename: Yizhi
authors.initials: Y
authors.lastname: Cai
authors.affiliation: BGI-Shenzhen, Shenzhen 518083, China;
authors.forename: Tai
authors.initials: T
authors.lastname: Chen
authors.affiliation: High-Throughput Biology Center, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, USA;
authors.forename: Jessica S
authors.initials: JS
authors.lastname: Dymond
authors.affiliation: BGI-Shenzhen, Shenzhen 518083, China;
authors.forename: Kang
authors.initials: K
authors.lastname: Kang
authors.affiliation: BGI-Shenzhen, Shenzhen 518083, China;
authors.forename: Jianhui
authors.initials: J
authors.lastname: Gong
authors.affiliation: BGI-Shenzhen, Shenzhen 518083, China;
authors.forename: Xiaofan
authors.initials: X
authors.lastname: Zeng
authors.affiliation: BGI-Shenzhen, Shenzhen 518083, China;
authors.forename: Yongfen
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: BGI-Shenzhen, Shenzhen 518083, China;
authors.forename: Yingrui
authors.initials: Y
authors.lastname: Li
authors.affiliation: BGI-Shenzhen, Shenzhen 518083, China;
authors.forename: Qiang
authors.initials: Q
authors.lastname: Feng
authors.affiliation: BGI-Shenzhen, Shenzhen 518083, China;
authors.forename: Xun
authors.initials: X
authors.lastname: Xu
authors.affiliation: BGI-Shenzhen, Shenzhen 518083, China; Department of Biology, University of Copenhagen, DK-2200 Copenhagen, Denmark; Princess Al Jawhara Center of Excellence in the Research of Hereditary Disorders, King Abdulaziz University, Jeddah 21589, Saudi Arabia;
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.affiliation: BGI-Shenzhen, Shenzhen 518083, China;
authors.forename: Jian
authors.initials: J
authors.lastname: Wang
authors.affiliation: BGI-Shenzhen, Shenzhen 518083, China; James D. Watson Institute of Genome Science, Hangzhou 310058, China.
authors.forename: Huanming
authors.initials: H
authors.lastname: Yang
authors.affiliation: Department of Biochemistry and Molecular Pharmacology and Institute for Systems Genetics, NYU Langone Medical Center, New York, New York 10016, USA;
authors.forename: Jef D
authors.initials: JD
authors.lastname: Boeke
authors.affiliation: High-Throughput Biology Center, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, USA; Department of Biomedical Engineering, School of Engineering, Johns Hopkins University, Baltimore, Maryland 21218, USA;
authors.forename: Joel S
authors.initials: JS
authors.lastname: Bader
mesh_terms: D058674:Chromosome Duplication; D007446:Chromosome Inversion; D002875:Chromosomes / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics; D019020:Directed Molecular Evolution; D015321:Gene Rearrangement; D016681:Genome, Fungal; D059014:High-Throughput Nucleotide Sequencing; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion
chemical_list: D004271:DNA, Fungal