Epilepsy in ring 14 chromosome syndrome

Specchio, Nicola; Trivisano, Marina; Serino, Domenico; Cappelletti, Simona; Carotenuto, Antonio; Claps, Dianela; Marras, Carlo Efisio; Fusco, Lucia; Elia, Maurizio; Vigevano, Federico

doi:10.1016/j.yebeh.2012.09.032

Cited by 16 publications

(9 citation statements)

References 23 publications

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“…We converted a linear synV to a ring by design, a process that could eventually enable more precise modeling of ring chromosome disorders if performed in mammalian cells. Circular "ring" chromosomes have been reported in a wide variety of human genetic disorders, including epilepsy (14,15), intellectual delay (15), various dysmorphic features (16), leukemia (17), and microcephaly (18,19). Further, therapies for genetic disorders based on chromosome circularization have been proposed (20).…”

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confidence: 99%

“Perfect” designer chromosome V and behavior of a ring derivative

Xie

Mitchell

et al. 2017

Science

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INTRODUCTION The Saccharomyces cerevisiae 2.0 project (Sc2.0) aims to modify the yeast genome with a series of densely spaced designer changes. Both a synthetic yeast chromosome arm (synIXR) and the entirely synthetic chromosome (synIII) function with high fitness in yeast. For designer genome synthesis projects, precise engineering of the physical sequence to match the specified design is important for the systematic evaluation of underlying design principles. Yeast can maintain nuclear chromosomes as rings, occurring by chance at repeated sequences, although the cyclized format is unfavorable in meiosis given the possibility of dicentric chromosome formation from meiotic recombination. Here, we describe the de novo synthesis of synthetic yeast chromosome V (synV) in the “Build-A-Genome China” course, perfectly matching the designer sequence and bearing loxPsym sites, distinguishable watermarks, and all the other features of the synthetic genome. We generated a ring synV derivative with user-specified cyclization coordinates and characterized its performance in mitosis and meiosis. RATIONALE Systematic evaluation of underlying Sc2.0 design principles requires that the final assembled synthetic genome perfectly match the designed sequence. Given the size of yeast chromosomes, synthetic chromosome construction is performed iteratively, and new mutations and unpredictable events may occur during synthesis; even a very small number of unintentional nucleotide changes across the genome could have substantial effects on phenotype. Therefore, precisely matching the physical sequence to the designed sequence is crucial for verification of the design principles in genome synthesis. Ring chromosomes can extend those design principles to provide a model for genomic rearrangement, ring chromosome evolution, and human ring chromosome disorders. RESULTS We chemically synthesized, assembled, and incorporated designer chromosome synV (536,024 base pairs) of S. cerevisiae according to Sc2.0 principles, based on the complete nucleotide sequence of native yeast chromosome V (576,874 base pairs). This work was performed as part of the “Build-A-Genome China” course in Tianjin University. We corrected all mutations found—including duplications, substitutions, and indels—in the initial synV strain by using integrative cotransformation of the precise desired changes and by means of a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)–based method. Altogether, 3331 corrected base pairs were required to match to the designed sequence. We generated a strain that exactly matches all designer sequence changes that displays high fitness under a variety of culture conditions. All corrections were verified with whole-genome sequencing; RNA sequencing revealed only minor changes in gene expression—most notably, decreases in expression of genes relocated near synthetic telomeres as a result of design. We constructed a functional circular synV (ring_synV) derivative in yeast by precisely joining both chromosome ends (telomeres) at specified coordinates. The ring chromosome showed restoration of subtelomeric gene expression levels. The ring_synV strain exhibited fitness comparable with that of the linear synV strain, revealed no change in sporulation frequency, but notably reduced spore viability. In meiosis, heterozygous or homozygous diploid ring_wtV and ring_synV chromosomes behaved similarly, exhibiting substantially higher frequency of the formation of zero-spore tetrads, a type that was not seen in the rod chromosome diploids. Rod synV chromosomes went through meiosis with high spore viability, despite no effort having been made to preserve meiotic competency in the design of synV. CONCLUSION The perfect designer-matched synthetic chromosome V provides strategies to edit sequence variants and correct unpredictable events, such as off-target integration of extra copies of synthetic DNA elsewhere in the genome. We also constructed a ring synthetic chromosome derivative and evaluated its fitness and stability in yeast. Both synV and synVI can be circularized and can power yeast cell growth without affecting fitness when gene content is maintained. These fitness and stability phenotypes of the ring synthetic chromosome in yeast provide a model system with which to probe the mechanism of human ring chromosome disorders. Synthesis, cyclization, and characterization of synV . ( A ) Synthetic chromosome V (synV, 536,024 base pairs) was designed in silico from native chromosome V (wtV, 576,874 base pairs), with extensive genotype modification designed to be phenotypically neutral. ( B ) CRISPR/Cas9 strategy for multiplex repair. ( C ) Colonies of wtV, synV, and ring_synV strains.

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confidence: 99%

“Perfect” designer chromosome V and behavior of a ring derivative

Xie

Mitchell

et al. 2017

Science

205

151

View full text Add to dashboard Cite

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“…Ring chromosomes have been found for nearly all human chromosomes and can be generated through different mechanisms, such as breaks in the chromosome arms followed by fusion of the proximal broken ends, fusion of two subtelomeric regions, or telomere–telomere fusion, among others. In addition, ring instability triggers secondary aberrations, including the loss or gain of genetic material, as well as other structural conformations, resulting in highly variable syndromes, for example, epilepsy, intellectual disabilities, leukemia, microcephaly, and others . Considering the complexity of inheritance and the pleiotropy associated with human ring chromosomes, the ability to create a stable and modifiable yeast ring synV chromosome in which changes can be tracked might provide an alternative model for exploring the mechanisms of disorders associated with ring chromosomes.…”

Section: Application Of Synthesized Genomes For Disease Researchmentioning

confidence: 99%

Synthetic Genomics: From DNA Synthesis to Genome Design

et al. 2018

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Rapid technological advances enabling the construction of designer gene networks, biosynthetic pathways, and even entire genomes are moving the fields of genetics and genomics from descriptive to synthetic applications. Following the synthesis of small viral genomes, advances in DNA assembly and rewriting have enabled the hierarchical synthesis of bacterial genomes, such as Mycoplasma genitalium, as well as recoding of the Escherichia coli genome by reducing the number of codons from 64 to 57. The field has advanced to the point of synthesizing an entire eukaryotic genome. The Synthetic Yeast Genome Project (Sc2.0) is underway and aims to rewrite all 16 Saccharomyces cerevisiae chromosomes by 2018; to date, 6.5 chromosomes have been designed and synthesized. Using bottom-up assembly and applying genome-wide alterations will improve our understanding of genome structure and function. This approach will not only provide a platform for systematic studies of eukaryotic chromosomes but will also generate diverse "streamlined" strains that are potentially suitable for medical and industrial applications. Herein, we review the current state of synthetic genome research and discuss potential applications of this emerging technology.

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“…Xie [43] geistige Behinderungen, [43b] Leukämie, [44] Mikrozephalie [45] und andere [46] hervorgehen kçnnen. Darüber hinaus ermçglicht die synthetische Genomik die strukturelle Modifizierung von Chromosomen.…”

Section: Anwendung Synthetischer Genome In Der Medizinischen Forschungunclassified

Synthetische Genomik: von der DNA‐Synthese zu Designer‐Genomen

et al. 2018

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Rasante technische Fortschritte, die die Konstruktion von genetischen Netzwerken, Biosynthesepfaden und sogar ganzer Genome ermöglichen, konvertieren derzeit die Genetik und Genomik von rein deskriptiven zu synthetischen Wissenschaften. Anknüpfend an die Synthese kleiner viraler Genome haben Fortschritte bei der Assemblierung und Neusynthese von DNA die hierarchische Synthese bakterieller Genome, z. B. von Mycoplasma genitalium, sowie die Umkodierung des Genoms von Escherichia coli durch Reduzierung der Zahl an Codons von 64 auf 57 ermöglicht. Das Gebiet ist an dem Punkt angekommen, dass wir ein gesamtes eukaryotisches Genom synthetisieren können. Das “Synthetic Yeast Genome Project” (Sc2.0) folgt den Plan, bis 2018 alle 16 Saccharomyces cerevisiae‐Chromosomen neu zu synthetisieren; bisher wurden 6.5 Chromosomen konzipiert und synthetisiert. Bottom‐up‐Assemblierung und die Anwendung genomweiter Modifikationen werden unser Verständnis der Genomstruktur und ‐funktion verbessern. Dieser Ansatz wird nicht nur eine Plattform für die systemische Erforschung eukaryotischer Chromosomen bieten, sondern auch “verschlankte” Stämme hervorbringen, die für medizinische und industrielle Anwendungen potenziell geeignet sind. In diesem Kurzaufsatz fassen wir den aktuellen Stand der Forschung im Bereich synthetischer Genome zusammen und diskutieren mögliche Anwendungen dieser neuen Technologie.

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Epilepsy in ring 14 chromosome syndrome

Cited by 16 publications

References 23 publications

“Perfect” designer chromosome V and behavior of a ring derivative

“Perfect” designer chromosome V and behavior of a ring derivative

Synthetic Genomics: From DNA Synthesis to Genome Design

Synthetische Genomik: von der DNA‐Synthese zu Designer‐Genomen

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