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1.
Nature ; 548(7668): 413-419, 2017 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-28783728

RESUMO

Genome editing has potential for the targeted correction of germline mutations. Here we describe the correction of the heterozygous MYBPC3 mutation in human preimplantation embryos with precise CRISPR-Cas9-based targeting accuracy and high homology-directed repair efficiency by activating an endogenous, germline-specific DNA repair response. Induced double-strand breaks (DSBs) at the mutant paternal allele were predominantly repaired using the homologous wild-type maternal gene instead of a synthetic DNA template. By modulating the cell cycle stage at which the DSB was induced, we were able to avoid mosaicism in cleaving embryos and achieve a high yield of homozygous embryos carrying the wild-type MYBPC3 gene without evidence of off-target mutations. The efficiency, accuracy and safety of the approach presented suggest that it has potential to be used for the correction of heritable mutations in human embryos by complementing preimplantation genetic diagnosis. However, much remains to be considered before clinical applications, including the reproducibility of the technique with other heterozygous mutations.


Assuntos
Proteínas de Transporte/genética , Embrião de Mamíferos/metabolismo , Edição de Genes/métodos , Mutação/genética , Adulto , Alelos , Blastocisto/metabolismo , Blastocisto/patologia , Divisão Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Quebras de DNA de Cadeia Dupla , Embrião de Mamíferos/patologia , Marcação de Genes , Teste de Complementação Genética , Heterozigoto , Homozigoto , Humanos , Masculino , Mosaicismo , Reparo de DNA por Recombinação/genética , Fase S , Moldes Genéticos , Zigoto/metabolismo , Zigoto/patologia
2.
Genome Res ; 26(1): 36-49, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26566658

RESUMO

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.


Assuntos
Cromossomos/genética , Evolução Molecular Direcionada , Rearranjo Gênico , Genoma Fúngico , Duplicação Cromossômica , Inversão Cromossômica , DNA Fúngico/genética , Sequenciamento de Nucleotídeos em Larga Escala , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA , Deleção de Sequência
4.
Sensors (Basel) ; 19(10)2019 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-31109014

RESUMO

A regenerated fiber Bragg grating (RFBG) in silica fiber was used to observe the viscous relaxation process of the host silica fiber at high temperatures of around 1000 °C. Two factors, preannealing time and loaded tension, which affect viscous relaxation, were observed. When an RFBG is stretched after a longer preannealing, the measured viscosity of the optical fiber was observed to reach equilibrium faster, which means that preannealing accelerates viscous relaxation. A similar acceleration phenomenon was also observed when a larger load was applied to stretch the optical fiber, although the acceleration effect of loaded tension was not as strong as in the preannealing case. The results play an active role in establishing effective optical-fiber devices for application in high-temperature environments.

5.
Cell Genom ; 3(11): 100364, 2023 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-38020968

RESUMO

Aneuploidy compromises genomic stability, often leading to embryo inviability, and is frequently associated with tumorigenesis and aging. Different aneuploid chromosome stoichiometries lead to distinct transcriptomic and phenotypic changes, making it helpful to study aneuploidy in tightly controlled genetic backgrounds. By deploying the engineered SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution) system to the newly synthesized megabase Sc2.0 chromosome VII (synVII), we constructed a synthetic disomic yeast and screened hundreds of SCRaMbLEd derivatives with diverse chromosomal rearrangements. Phenotypic characterization and multi-omics analysis revealed that fitness defects associated with aneuploidy could be restored by (1) removing most of the chromosome content or (2) modifying specific regions in the duplicated chromosome. These findings indicate that both chromosome copy number and specific chromosomal regions contribute to the aneuploidy-related phenotypes, and the synthetic chromosome resource opens new paradigms in studying aneuploidy.

6.
Nat Commun ; 14(1): 1219, 2023 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-36882397

RESUMO

Range of DNA repair in response to double-strand breaks induced in human preimplantation embryos remains uncertain due to the complexity of analyzing single- or few-cell samples. Sequencing of such minute DNA input requires a whole genome amplification that can introduce artifacts, including coverage nonuniformity, amplification biases, and allelic dropouts at the target site. We show here that, on average, 26.6% of preexisting heterozygous loci in control single blastomere samples appear as homozygous after whole genome amplification indicative of allelic dropouts. To overcome these limitations, we validate on-target modifications seen in gene edited human embryos in embryonic stem cells. We show that, in addition to frequent indel mutations, biallelic double-strand breaks can also produce large deletions at the target site. Moreover, some embryonic stem cells show copy-neutral loss of heterozygosity at the cleavage site which is likely caused by interallelic gene conversion. However, the frequency of loss of heterozygosity in embryonic stem cells is lower than in blastomeres, suggesting that allelic dropouts is a common whole genome amplification outcome limiting genotyping accuracy in human preimplantation embryos.


Assuntos
Blastocisto , Edição de Genes , Humanos , Blastômeros , Embrião de Mamíferos , Alelos
7.
Cell Res ; 29(10): 804-819, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31444470

RESUMO

In vivo genome editing represents a powerful strategy for both understanding basic biology and treating inherited diseases. However, it remains a challenge to develop universal and efficient in vivo genome-editing tools for tissues that comprise diverse cell types in either a dividing or non-dividing state. Here, we describe a versatile in vivo gene knock-in methodology that enables the targeting of a broad range of mutations and cell types through the insertion of a minigene at an intron of the target gene locus using an intracellularly linearized single homology arm donor. As a proof-of-concept, we focused on a mouse model of premature-aging caused by a dominant point mutation, which is difficult to repair using existing in vivo genome-editing tools. Systemic treatment using our new method ameliorated aging-associated phenotypes and extended animal lifespan, thus highlighting the potential of this methodology for a broad range of in vivo genome-editing applications.


Assuntos
Edição de Genes/métodos , Animais , Sistemas CRISPR-Cas/genética , Reparo do DNA , Dependovirus/genética , Fator de Transcrição GATA3/genética , Técnicas de Introdução de Genes , Terapia Genética/métodos , Vetores Genéticos/metabolismo , Células-Tronco Embrionárias Humanas , Humanos , Íntrons , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Neurônios/citologia , Neurônios/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , Ratos , Tubulina (Proteína)/genética
8.
G3 (Bethesda) ; 7(12): 3857-3866, 2017 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-29038170

RESUMO

Using a comprehensive library of histone H2A and H2B mutants, we assessed the biological function of each amino acid residue involved in various stress conditions including exposure to different DNA damage-inducing reagents, different growth temperatures, and other chemicals. H2B N- and H2A C-termini were critical for maintaining nucleosome function and mutations in these regions led to pleiotropic phenotypes. Additionally, two screens were performed using this library, monitoring heterochromatin gene silencing and genome stability, to identify residues that could compromise normal function when mutated. Many distinctive regions within the nucleosome were revealed. Furthermore, we used the barcode sequencing (bar-seq) method to profile the mutant composition of many libraries in one high-throughput sequencing experiment, greatly reducing the labor and increasing the capacity. This study not only demonstrates the applications of the versatile histone library, but also reveals many previously unknown functions of histone H2A and H2B.


Assuntos
Dano ao DNA/efeitos dos fármacos , Histonas/genética , Nucleossomos/genética , Aminoácidos/genética , Biblioteca Gênica , Inativação Gênica , Instabilidade Genômica/genética , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas Mutantes/genética , Ligação Proteica , Saccharomyces cerevisiae/genética
9.
Science ; 355(6329)2017 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-28280149

RESUMO

We designed and synthesized a 976,067-base pair linear chromosome, synXII, based on native chromosome XII in Saccharomyces cerevisiae SynXII was assembled using a two-step method, specified by successive megachunk integration and meiotic recombination-mediated assembly, producing a functional chromosome in S. cerevisiae. Minor growth defect "bugs" detected in synXII, caused by deletion of tRNA genes, were rescued by introducing an ectopic copy of a single tRNA gene. The ribosomal gene cluster (rDNA) on synXII was left intact during the assembly process and subsequently replaced by a modified rDNA unit used to regenerate rDNA at three distinct chromosomal locations. The signature sequences within rDNA, which can be used to determine species identity, were swapped to generate a Saccharomyces synXII strain that would be identified as Saccharomyces bayanus by standard DNA barcoding procedures.


Assuntos
Cromossomos Artificiais de Levedura/química , DNA Ribossômico/genética , Engenharia Genética/métodos , Genoma Fúngico , Saccharomyces cerevisiae/genética , Biologia Sintética/métodos , Núcleo Celular/genética , Núcleo Celular/ultraestrutura , Cromossomos Artificiais de Levedura/genética , Cromossomos Artificiais de Levedura/ultraestrutura , Saccharomyces cerevisiae/ultraestrutura , Transcriptoma
10.
Science ; 355(6329)2017 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-28280153

RESUMO

Here, we report the successful design, construction, and characterization of a 770-kilobase synthetic yeast chromosome II (synII). Our study incorporates characterization at multiple levels-including phenomics, transcriptomics, proteomics, chromosome segregation, and replication analysis-to provide a thorough and comprehensive analysis of a synthetic chromosome. Our Trans-Omics analyses reveal a modest but potentially relevant pervasive up-regulation of translational machinery observed in synII, mainly caused by the deletion of 13 transfer RNAs. By both complementation assays and SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution), we targeted and debugged the origin of a growth defect at 37°C in glycerol medium, which is related to misregulation of the high-osmolarity glycerol response. Despite the subtle differences, the synII strain shows highly consistent biological processes comparable to the native strain.


Assuntos
Cromossomos Artificiais de Levedura/fisiologia , Genoma Fúngico , Saccharomyces cerevisiae/genética , Segregação de Cromossomos , Cromossomos Artificiais de Levedura/química , Cromossomos Artificiais de Levedura/genética , Meios de Cultura/química , Replicação do DNA , Glicerol , Proteômica , Saccharomyces cerevisiae/crescimento & desenvolvimento , Análise de Sequência de DNA , Biologia Sintética , Transcriptoma
11.
Sci Rep ; 5: 14465, 2015 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-26403826

RESUMO

The forfeiting of photosynthetic capabilities has occurred independently many times throughout eukaryotic evolution. But almost all non-photosynthetic plants and algae still retain a colorless plastid and an associated genome, which performs fundamental processes apart from photosynthesis. Unfortunately, little is known about the forces leading to photosynthetic loss; this is largely because there is a lack of data from transitional species. Here, we compare the plastid genomes of two "transitional" green algae: the photosynthetic, mixotrophic Auxenochlorella protothecoides and the non-photosynthetic, obligate heterotroph Prototheca wickerhamii. Remarkably, the plastid genome of A. protothecoides is only slightly larger than that of P. wickerhamii, making it among the smallest plastid genomes yet observed from photosynthetic green algae. Even more surprising, both algae have almost identical plastid genomic architectures and gene compositions (with the exception of genes involved in photosynthesis), implying that they are closely related. This close relationship was further supported by phylogenetic and substitution rate analyses, which suggest that the lineages giving rise to A. protothecoides and P. wickerhamii diverged from one another around six million years ago.


Assuntos
Clorófitas/genética , Genomas de Plastídeos , Genômica , Prototheca/genética , Clorófitas/metabolismo , Ordem dos Genes , Mutação , Taxa de Mutação , Fotossíntese/genética , Filogenia , Prototheca/metabolismo
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