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We designed and synthesized synI, which is â¼21.6% shorter than native chrI, the smallest chromosome in Saccharomyces cerevisiae. SynI was designed for attachment to another synthetic chromosome due to concerns surrounding potential instability and karyotype imbalance and is now attached to synIII, yielding the first synthetic yeast fusion chromosome. Additional fusion chromosomes were constructed to study nuclear function. ChrIII-I and chrIX-III-I fusion chromosomes have twisted structures, which depend on silencing protein Sir3. As a smaller chromosome, chrI also faces special challenges in assuring meiotic crossovers required for efficient homolog disjunction. Centromere deletions into fusion chromosomes revealed opposing effects of core centromeres and pericentromeres in modulating deposition of the crossover-promoting protein Red1. These effects extend over 100 kb and promote disproportionate Red1 enrichment, and thus crossover potential, on small chromosomes like chrI. These findings reveal the power of synthetic genomics to uncover new biology and deconvolute complex biological systems.
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The Sc2.0 project is building a eukaryotic synthetic genome from scratch. A major milestone has been achieved with all individual Sc2.0 chromosomes assembled. Here, we describe the consolidation of multiple synthetic chromosomes using advanced endoreduplication intercrossing with tRNA expression cassettes to generate a strain with 6.5 synthetic chromosomes. The 3D chromosome organization and transcript isoform profiles were evaluated using Hi-C and long-read direct RNA sequencing. We developed CRISPR Directed Biallelic URA3-assisted Genome Scan, or "CRISPR D-BUGS," to map phenotypic variants caused by specific designer modifications, known as "bugs." We first fine-mapped a bug in synthetic chromosome II (synII) and then discovered a combinatorial interaction associated with synIII and synX, revealing an unexpected genetic interaction that links transcriptional regulation, inositol metabolism, and tRNASerCGA abundance. Finally, to expedite consolidation, we employed chromosome substitution to incorporate the largest chromosome (synIV), thereby consolidating >50% of the Sc2.0 genome in one strain.
Assuntos
Cromossomos Artificiais de Levedura , Genoma Fúngico , Saccharomyces cerevisiae , Sequência de Bases , Cromossomos/genética , Saccharomyces cerevisiae/genética , Biologia SintéticaRESUMO
The widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease derives its DNA targeting specificity from protein-DNA contacts with protospacer adjacent motif (PAM) sequences, in addition to base-pairing interactions between its guide RNA and target DNA. Previous reports have established that the PAM specificity of SpCas9 can be altered via positive selection procedures for directed evolution or other protein engineering strategies. Here we exploit in vivo directed evolution systems that incorporate simultaneous positive and negative selection to evolve SpCas9 variants with commensurate or improved activity on NAG PAMs relative to wild type and reduced activity on NGG PAMs, particularly YGG PAMs. We also show that the PAM preferences of available evolutionary intermediates effectively determine whether similar counterselection PAMs elicit different selection stringencies, and demonstrate that negative selection can be specifically increased in a yeast selection system through the fusion of compensatory zinc fingers to SpCas9.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , DNA/metabolismo , Edição de Genes/métodos , RNA Guia de Cinetoplastídeos/metabolismo , Streptococcus pyogenes/metabolismo , Sequência de Aminoácidos , Proteína 9 Associada à CRISPR/genética , Linhagem Celular Tumoral , DNA/química , DNA/genética , Evolução Molecular Direcionada/métodos , Humanos , Mutação , Conformação de Ácido Nucleico , Motivos de Nucleotídeos/genética , Engenharia de Proteínas/métodos , RNA Guia de Cinetoplastídeos/genética , Streptococcus pyogenes/genética , Especificidade por SubstratoRESUMO
Long interspersed element-1 (LINE-1, or L1) is the only autonomous retrotransposon that is active in human cells. Different host factors have been shown to influence L1 mobility; however, systematic analyses of these factors are limited. Here, we developed a high-throughput microscopy-based retrotransposition assay that identified the double-stranded break (DSB) repair and Fanconi anemia (FA) factors active in the S/G2 phase as potent inhibitors and regulators of L1 activity. In particular, BRCA1, an E3 ubiquitin ligase with a key role in several DNA repair pathways, directly affects L1 retrotransposition frequency and structure and plays a distinct role in controlling L1 ORF2 protein translation through L1 mRNA binding. These results suggest the existence of a 'battleground' at the DNA replication fork between homologous recombination (HR) factors and L1 retrotransposons and reveal a potential role for L1 in the genotypic evolution of tumors characterized by BRCA1 and HR repair deficiencies.
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Proteína BRCA1/metabolismo , Reparo do DNA , Elementos Nucleotídeos Longos e Dispersos , Fase S , Proteína BRCA1/genética , Sistemas CRISPR-Cas , Linhagem Celular , Quebras de DNA de Cadeia Dupla , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Deleção de Genes , Células HEK293 , Células HeLa , Recombinação Homóloga , Humanos , MicroscopiaRESUMO
Cross-species pathway transplantation enables insight into a biological process not possible through traditional approaches. We replaced the enzymes catalyzing the entire Saccharomyces cerevisiae adenine de novo biosynthesis pathway with the human pathway. While the 'humanized' yeast grew in the absence of adenine, it did so poorly. Dissection of the phenotype revealed that PPAT, the human ortholog of ADE4, showed only partial function whereas all other genes complemented fully. Suppressor analysis revealed other pathways that play a role in adenine de-novo pathway regulation. Phylogenetic analysis pointed to adaptations of enzyme regulation to endogenous metabolite level 'setpoints' in diverse organisms. Using DNA shuffling, we isolated specific amino acids combinations that stabilize the human protein in yeast. Thus, using adenine de novo biosynthesis as a proof of concept, we suggest that the engineering methods used in this study as well as the debugging strategies can be utilized to transplant metabolic pathway from any origin into yeast.
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Adenina/biossíntese , Vias Biossintéticas/genética , Carboxiliases/genética , Cromossomos Artificiais Humanos/química , Peptídeo Sintases/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sistemas CRISPR-Cas , Carboxiliases/metabolismo , Cromossomos Artificiais Humanos/metabolismo , Teste de Complementação Genética , Engenharia Genética/métodos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Peptídeo Sintases/metabolismo , Filogenia , Plasmídeos/química , Plasmídeos/metabolismo , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The original version of this Article omitted a declaration from the Competing Interests statement, which should have included the following: 'J.D.B. is a founder and Director of the following: Neochromosome, Inc., the Center of Excellence for Engineering Biology, and CDI Labs, Inc. and serves on the Scientific Advisory Board of the following: Modern Meadow, Inc., Recombinetics, Inc., and Sample6, Inc.'. This has now been corrected in both the PDF and HTML versions of the Article.
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Engineering multicellularity is one of the next breakthroughs for Synthetic Biology. A key bottleneck to building multicellular systems is the lack of a scalable signaling language with a large number of interfaces that can be used simultaneously. Here, we present a modular, scalable, intercellular signaling language in yeast based on fungal mating peptide/G-protein-coupled receptor (GPCR) pairs harnessed from nature. First, through genome-mining, we assemble 32 functional peptide-GPCR signaling interfaces with a range of dose-response characteristics. Next, we demonstrate that these interfaces can be combined into two-cell communication links, which serve as assembly units for higher-order communication topologies. Finally, we show 56 functional, two-cell links, which we use to assemble three- to six-member communication topologies and a three-member interdependent community. Importantly, our peptide-GPCR language is scalable and tunable by genetic encoding, requires minimal component engineering, and should be massively scalable by further application of our genome mining pipeline or directed evolution.
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Peptídeos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Biologia Computacional/métodos , Peptídeos/genética , Ligação Proteica , Receptores Acoplados a Proteínas G/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Biologia Sintética/métodosRESUMO
Rapid and highly efficient mating-type switching of Saccharomyces cerevisiae enables a wide variety of genetic manipulations, such as the construction of strains, for instance, isogenic haploid pairs of both mating-types, diploids and polyploids. We used the CRISPR/Cas9 system to generate a double-strand break at the MAT locus and, in a single cotransformation, both haploid and diploid cells were switched to the specified mating-type at â¼80% efficiency. The mating-type of strains carrying either rod or ring chromosome III were switched, including those lacking HMLα and HMRa cryptic mating loci. Furthermore, we transplanted the synthetic yeast chromosome V to build a haploid polysynthetic chromosome strain by using this method together with an endoreduplication intercross strategy. The CRISPR/Cas9 mating-type switching method will be useful in building the complete synthetic yeast (Sc2.0) genome. Importantly, it is a generally useful method to build polyploids of a defined genotype and generally expedites strain construction, for example, in the construction of fully a/a/α/α isogenic tetraploids.
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Sistemas CRISPR-Cas , DNA Fúngico/genética , Edição de Genes/métodos , Genes Fúngicos Tipo Acasalamento , Genoma Fúngico , Saccharomyces cerevisiae/genética , Engenharia Celular/métodos , Cromossomos Artificiais/química , Quebras de DNA de Cadeia Dupla , DNA Fúngico/metabolismo , Loci Gênicos , Plasmídeos/química , Plasmídeos/metabolismo , Ploidias , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
As the use of synthetic biology both in industry and in academia grows, there is an increasing need to ensure biocontainment. There is growing interest in engineering bacterial- and yeast-based safeguard (SG) strains. First-generation SGs were based on metabolic auxotrophy; however, the risk of cross-feeding and the cost of growth-controlling nutrients led researchers to look for other avenues. Recent strategies include bacteria engineered to be dependent on nonnatural amino acids and yeast SG strains that have both transcriptional- and recombinational-based biocontainment. We describe improving yeast Saccharomyces cerevisiae-based transcriptional SG strains, which have near-WT fitness, the lowest possible escape rate, and nanomolar ligands controlling growth. We screened a library of essential genes, as well as the best-performing promoter and terminators, yielding the best SG strains in yeast. The best constructs were fine-tuned, resulting in two tightly controlled inducible systems. In addition, for potential use in the prevention of industrial espionage, we screened an array of possible "decoy molecules" that can be used to mask any proprietary supplement to the SG strain, with minimal effect on strain fitness.
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Genoma/genética , Saccharomyces cerevisiae/genética , Genes Essenciais/genética , Engenharia Metabólica/métodos , Regiões Promotoras Genéticas/genética , Biologia Sintética/métodos , Regiões Terminadoras Genéticas/genética , Transcrição Gênica/genéticaRESUMO
We have developed a method for assembling genetic pathways for expression in Saccharomyces cerevisiae. Our pathway assembly method, called VEGAS (Versatile genetic assembly system), exploits the native capacity of S. cerevisiae to perform homologous recombination and efficiently join sequences with terminal homology. In the VEGAS workflow, terminal homology between adjacent pathway genes and the assembly vector is encoded by 'VEGAS adapter' (VA) sequences, which are orthogonal in sequence with respect to the yeast genome. Prior to pathway assembly by VEGAS in S. cerevisiae, each gene is assigned an appropriate pair of VAs and assembled using a previously described technique called yeast Golden Gate (yGG). Here we describe the application of yGG specifically to building transcription units for VEGAS assembly as well as the VEGAS methodology. We demonstrate the assembly of four-, five- and six-gene pathways by VEGAS to generate S. cerevisiae cells synthesizing ß-carotene and violacein. Moreover, we demonstrate the capacity of yGG coupled to VEGAS for combinatorial assembly.
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Vias Biossintéticas/genética , Saccharomyces cerevisiae/genética , Genes Fúngicos , Vetores Genéticos , Recombinação Homóloga , Indóis/metabolismo , Reação em Cadeia da Polimerase , Biologia Sintética/métodos , Transcrição Gênica , beta Caroteno/biossínteseRESUMO
We have adapted the Golden Gate DNA assembly method to the assembly of transcription units (TUs) for the yeast Saccharomyces cerevisiae, in a method we call yeast Golden Gate (yGG). yGG allows for the easy assembly of TUs consisting of promoters (PRO), coding sequences (CDS), and terminators (TER). Carefully designed overhangs exposed by digestion with a type IIS restriction enzyme enable virtually seamless assembly of TUs that, in principle, contain all of the information necessary to express a gene of interest in yeast. We also describe a versatile set of yGG acceptor vectors to be used for TU assembly. These vectors can be used for low or high copy expression of assembled TUs or integration into carefully selected innocuous genomic loci. yGG provides synthetic biologists and yeast geneticists with an efficient new means by which to engineer S. cerevisiae.
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Saccharomyces cerevisiae/metabolismo , Regiões 3' não Traduzidas , DNA/genética , DNA/metabolismo , Engenharia Genética , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Fases de Leitura Aberta/genética , Regiões Promotoras GenéticasRESUMO
Biocontainment may be required in a wide variety of situations such as work with pathogens, field release applications of engineered organisms, and protection of intellectual properties. Here, we describe the control of growth of the brewer's yeast, Saccharomyces cerevisiae, using both transcriptional and recombinational "safeguard" control of essential gene function. Practical biocontainment strategies dependent on the presence of small molecules require them to be active at very low concentrations, rendering them inexpensive and difficult to detect. Histone genes were controlled by an inducible promoter and controlled by 30 nM estradiol. The stability of the engineered genes was separately regulated by the expression of a site-specific recombinase. The combined frequency of generating viable derivatives when both systems were active was below detection (<10(-10)), consistent with their orthogonal nature and the individual escape frequencies of <10(-6). Evaluation of escaper mutants suggests strategies for reducing their emergence. Transcript profiling and growth test suggest high fitness of safeguarded strains, an important characteristic for wide acceptance.
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Biotecnologia/métodos , Contenção de Riscos Biológicos/métodos , Genes Essenciais/genética , Engenharia Genética/métodos , Organismos Geneticamente Modificados/genética , Saccharomyces cerevisiae/genética , Perfilação da Expressão Gênica , Mutação/genética , Recombinases/metabolismo , Recombinação Genética/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transcrição Gênica/genéticaRESUMO
Rapid advances in DNA synthesis techniques have made it possible to engineer viruses, biochemical pathways and assemble bacterial genomes. Here, we report the synthesis of a functional 272,871-base pair designer eukaryotic chromosome, synIII, which is based on the 316,617-base pair native Saccharomyces cerevisiae chromosome III. Changes to synIII include TAG/TAA stop-codon replacements, deletion of subtelomeric regions, introns, transfer RNAs, transposons, and silent mating loci as well as insertion of loxPsym sites to enable genome scrambling. SynIII is functional in S. cerevisiae. Scrambling of the chromosome in a heterozygous diploid reveals a large increase in a-mater derivatives resulting from loss of the MATα allele on synIII. The complete design and synthesis of synIII establishes S. cerevisiae as the basis for designer eukaryotic genome biology.
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Cromossomos Fúngicos , Saccharomyces cerevisiae/genética , Biologia Sintética/métodos , Sequência de Bases , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/metabolismo , DNA Fúngico/genética , Genes Fúngicos , Aptidão Genética , Genoma Fúngico , Instabilidade Genômica , Íntrons , Dados de Sequência Molecular , Mutação , Reação em Cadeia da Polimerase , RNA Fúngico/genética , RNA de Transferência/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologia , Análise de Sequência de DNA , Deleção de Sequência , Transformação GenéticaRESUMO
The most dangerous insults to the genome's integrity are those that break both strands of the DNA. Double-strand breaks can be repaired by homologous recombination; in this conserved mechanism, a global genomic homology search finds sequences similar to those near the break, and uses them as a template for DNA synthesis and ligation. Chromosomes occupy restricted territories within the nucleus. We show that yeast genomic regions whose nuclear territories overlap recombine more efficiently than sequences located in spatially distant territories. Tethering of telomeres and centromeres reduces the efficiency of recombination between distant genomic loci, lowering the chances of non-allelic recombination. Our results challenge present models that posit an active scanning of the whole nuclear volume by the broken chromosomal end; they demonstrate that the search for homology is a limiting step in homologous recombination, and emphasize the importance of nuclear organization in genome maintenance.
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Núcleo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA Fúngico/metabolismo , Saccharomyces cerevisiae/genética , Núcleo Celular/ultraestrutura , Centrômero/genética , Centrômero/metabolismo , Cromossomos Fúngicos , Dano ao DNA , DNA Fúngico/genética , Recombinação Homóloga , Recombinação Genética , Saccharomyces cerevisiae/ultraestrutura , Telômero/genética , Telômero/metabolismoRESUMO
DNA double-strand breaks (DSBs) and other lesions occur frequently during cell growth and in meiosis. These are often repaired by homologous recombination (HR). HR may result in the formation of DNA structures called Holliday junctions (HJs), which need to be resolved to allow chromosome segregation. Whereas HJs are present in most HR events in meiosis, it has been proposed that in vegetative cells most HR events occur through intermediates lacking HJs. A recent screen in yeast has shown HJ resolution activity for a protein called Yen1, in addition to the previously known Mus81/Mms4 complex. Yeast strains deleted for both YEN1 and MMS4 show a reduction in growth rate, and are very sensitive to DNA-damaging agents. In addition, we investigate the genetic interaction of yen1 and mms4 with mutants defective in different repair pathways. We find that in the absence of Yen1 and Mms4 deletion of RAD1 or RAD52 have no further effect, whereas additional sensitivity is seen if RAD51 is deleted. Finally, we show that yeast cells are unable to carry out meiosis in the absence of both resolvases. Our results show that both Yen1 and Mms4/Mus81 play important (although not identical) roles during vegetative growth and in meiosis.
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Dano ao DNA , Endonucleases Flap/fisiologia , Resolvases de Junção Holliday/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Endonucleases Flap/genética , Deleção de Genes , Resolvases de Junção Holliday/genética , Resolvases de Junção Holliday/metabolismo , Meiose , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
BACKGROUND: Extrachomosomal circular DNA (eccDNA) is ubiquitous in eukaryotic organisms and was detected in every organism tested, including in humans. A two-dimensional gel electrophoresis facilitates the detection of eccDNA in preparations of genomic DNA. Using this technique we have previously demonstrated that most of eccDNA consists of exact multiples of chromosomal tandemly repeated DNA, including both coding genes and satellite DNA. RESULTS: Here we report the occurrence of eccDNA in every tested human cell line. It has heterogeneous mass ranging from less than 2 kb to over 20 kb. We describe eccDNA homologous to human alpha satellite and the SstI mega satellite. Moreover, we show, for the first time, circular multimers of the human 5S ribosomal DNA (rDNA), similar to previous findings in Drosophila and plants. We further demonstrate structures that correspond to intermediates of rolling circle replication, which emerge from the circular multimers of 5S rDNA and SstI satellite. CONCLUSIONS: These findings, and previous reports, support the general notion that every chromosomal tandem repeat is prone to generate eccDNA in eukryoric organisms including humans. They suggest the possible involvement of eccDNA in the length variability observed in arrays of tandem repeats. The implications of eccDNA on genome biology may include mechanisms of centromere evolution, concerted evolution and homogenization of tandem repeats and genomic plasticity.
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To expand the known spectrum of genes that maintain genome stability, we screened a recently released collection of temperature sensitive (Ts) yeast mutants for a chromosome instability (CIN) phenotype. Proteasome subunit genes represented a major functional group, and subsequent analysis demonstrated an evolutionarily conserved role in CIN. Analysis of individual proteasome core and lid subunit mutations showed that the CIN phenotype at semi-permissive temperature is associated with failure of subunit localization to the nucleus. The resultant proteasome dysfunction affects chromosome stability by impairing the kinetics of double strand break (DSB) repair. We show that the DNA repair protein Mms22 is required for DSB repair, and recruited to chromatin in a ubiquitin-dependent manner as a result of DNA damage. Moreover, subsequent proteasome-mediated degradation of Mms22 is necessary and sufficient for cell cycle progression through the G(2)/M arrest induced by DNA damage. Our results demonstrate for the first time that a double strand break repair protein is a proteasome target, and thus link nuclear proteasomal activity and DSB repair.
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Núcleo Celular/enzimologia , Instabilidade Cromossômica , Reparo do DNA , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Alelos , Linhagem Celular , Cromatina/metabolismo , Proteínas Culina/metabolismo , Quebras de DNA de Cadeia Dupla , Regulação Fúngica da Expressão Gênica , Genes Essenciais , Humanos , Cinética , Mutação/genética , Ligação Proteica , Subunidades Proteicas/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/genética , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
Double-strand breaks (DSBs) occur frequently during cell growth. Due to the presence of repeated sequences in the genome, repair of a single DSB can result in gene conversion, translocation, deletion or tandem duplication depending on the mechanism and the sequence chosen as partner for the recombinational repair. Here, we study how yeast cells repair a single, inducible DSB when there are several potential donors to choose from, in the same chromosome and elsewhere in the genome. We systematically investigate the parameters that affect the choice of mechanism, as well as its genetic regulation. Our results indicate that intrachromosomal homologous sequences are always preferred as donors for repair. We demonstrate the occurrence of a novel tri-partite repair product that combines ectopic gene conversion and deletion. In addition, we show that increasing the distance between two repeated sequences enhances the dependence on Rad51 for colony formation after DSB repair. This is due to a role of Rad51 in the recovery from the checkpoint signal induced by the DSB. We suggest a model for the competition between the different homologous recombination pathways. Our model explains how different repair mechanisms are able to compensate for each other during DSB repair.
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Quebras de DNA de Cadeia Dupla , Reparo do DNA , Recombinação Genética , DNA Fúngico/química , Modelos Genéticos , Rad51 Recombinase/metabolismo , Sequências Repetitivas de Ácido Nucleico , Saccharomyces cerevisiae/genéticaRESUMO
Extrachromosomal circular DNA (eccDNA) is one characteristic of the plasticity of the eukaryotic genome. It is found in various organisms and contains sequences derived primarily from repetitive chromosomal DNA. Using 2D gel electrophoresis, we have previously detected eccDNA composed of chromosomal tandem repeats throughout the life cycle of Drosophila. Here, we report for the first time evidence suggesting the occurrence of rolling circle replication of eccDNA in Drosophila. We show, on 2D gels, specific structures that can be enriched by benzoylated naphthoylated DEAE-cellulose chromatography and were identified in other systems as rolling circle intermediates (RCIs). These RCIs are homologous to histone genes, Stellate and Suppressor of Stellate, which are all organized in the chromosomes as tandem repeats. RCIs are detected throughout the life cycle of Drosophila and in cultured fly cells. These structures are found regardless of the expression of the replicated gene or of its chromosomal copy number.