Synthetic chromosome fusion: Effects on mitotic and meiotic genome structure and function.

We designed and synthesized synI, which is ∼21.6% 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.

Context-dependent neocentromere activity in synthetic yeast chromosome VIII.

Pioneering advances in genome engineering, and specifically in genome writing, have revolutionized the field of synthetic biology, propelling us toward the creation of synthetic genomes. The Sc2.0 project aims to build the first fully synthetic eukaryotic organism by assembling the genome of Saccharomyces cerevisiae. With the completion of synthetic chromosome VIII (synVIII) described here, this goal is within reach. In addition to writing the yeast genome, we sought to manipulate an essential functional element: the point centromere. By relocating the native centromere sequence to various positions along chromosome VIII, we discovered that the minimal 118-bp CEN8 sequence is insufficient for conferring chromosomal stability at ectopic locations. Expanding the transplanted sequence to include a small segment (∼500 bp) of the CDEIII-proximal pericentromere improved chromosome stability, demonstrating that minimal centromeres display context-dependent functionality.

Translational fidelity screens in mammalian cells reveal eIF3 and eIF4G2 as regulators of start codon selectivity.

The translation initiation machinery and the ribosome orchestrate a highly dynamic scanning process to distinguish proper start codons from surrounding nucleotide sequences. Here, we performed genome-wide CRISPRi screens in human K562 cells to systematically identify modulators of the frequency of translation initiation at near-cognate start codons. We observed that depletion of any eIF3 core subunit promoted near-cognate start codon usage, though sensitivity thresholds of each subunit to sgRNA-mediated depletion varied considerably. Double sgRNA depletion experiments suggested that enhanced near-cognate usage in eIF3D depleted cells required canonical eIF4E cap-binding and was not driven by eIF2A or eIF2D-dependent leucine tRNA initiation. We further characterized the effects of eIF3D depletion and found that the N-terminus of eIF3D was strictly required for accurate start codon selection, whereas disruption of the cap-binding properties of eIF3D had no effect. Lastly, depletion of eIF3D activated TNFα signaling via NF-κB and the interferon gamma response. Similar transcriptional profiles were observed upon knockdown of eIF1A and eIF4G2, which also promoted near-cognate start codon usage, suggesting that enhanced near-cognate usage could potentially contribute to NF-κB activation. Our study thus provides new avenues to study the mechanisms and consequences of alternative start codon usage.

Two differentially stable rDNA loci coexist on the same chromosome and form a single nucleolus.

The nucleolus is the most prominent membraneless compartment within the nucleus-dedicated to the metabolism of ribosomal RNA. Nucleoli are composed of hundreds of ribosomal DNA (rDNA) repeated genes that form large chromosomal clusters, whose high recombination rates can cause nucleolar dysfunction and promote genome instability. Intriguingly, the evolving architecture of eukaryotic genomes appears to have favored two strategic rDNA locations-where a single locus per chromosome is situated either near the centromere (CEN) or the telomere. Here, we deployed an innovative genome engineering approach to cut and paste to an ectopic chromosomal location-the ~1.5 mega-base rDNA locus in a single step using CRISPR technology. This "megablock" rDNA engineering was performed in a fused-karyotype strain of Saccharomyces cerevisiae. The strategic repositioning of this locus within the megachromosome allowed experimentally mimicking and monitoring the outcome of an rDNA migratory event, in which twin rDNA loci coexist on the same chromosomal arm. We showed that the twin-rDNA yeast readily adapts, exhibiting wild-type growth and maintaining rRNA homeostasis, and that the twin loci form a single nucleolus throughout the cell cycle. Unexpectedly, the size of each rDNA array appears to depend on its position relative to the CEN, in that the locus that is CEN-distal undergoes size reduction at a higher frequency compared to the CEN-proximal counterpart. Finally, we provided molecular evidence supporting a mechanism called paralogous cis-rDNA interference, which potentially explains why placing two identical repeated arrays on the same chromosome may negatively affect their function and structural stability.

Karyotype engineering reveals spatio-temporal control of replication firing and gene contacts.

Eukaryotic genomes vary in terms of size, chromosome number, and genetic complexity. Their temporal organization is complex, reflecting coordination between DNA folding and function. Here, we used fused karyotypes of budding yeast to characterize the effects of chromosome length on nuclear architecture. We found that size-matched megachromosomes expand to occupy a larger fraction of the enlarged nucleus. Hi-C maps reveal changes in the three-dimensional structure corresponding to inactivated centromeres and telomeres. De-clustering of inactive centromeres results in their loss of early replication, highlighting a functional correlation between genome organization and replication timing. Repositioning of former telomere-proximal regions on chromosome arms exposed a subset of contacts between flocculin genes. Chromatin reorganization of megachromosomes during cell division remained unperturbed, and it revealed that centromere-rDNA contacts in anaphase, extending over 0.3 Mb on wild-type chromosome, cannot exceed ∼1.7 Mb. Our results highlight the relevance of engineered karyotypes to unveiling relationships between genome organization and function.

Karyotype engineering by chromosome fusion leads to reproductive isolation in yeast.

Extant species have wildly different numbers of chromosomes, even among taxa with relatively similar genome sizes (for example, insects)1,2. This is likely to reflect accidents of genome history, such as telomere-telomere fusions and genome duplication events3-5. Humans have 23 pairs of chromosomes, whereas other apes have 24. One human chromosome is a fusion product of the ancestral state6. This raises the question: how well can species tolerate a change in chromosome numbers without substantial changes to genome content? Many tools are used in chromosome engineering in Saccharomyces cerevisiae7-10, but CRISPR-Cas9-mediated genome editing facilitates the most aggressive engineering strategies. Here we successfully fused yeast chromosomes using CRISPR-Cas9, generating a near-isogenic series of strains with progressively fewer chromosomes ranging from sixteen to two. A strain carrying only two chromosomes of about six megabases each exhibited modest transcriptomic changes and grew without major defects. When we crossed a sixteen-chromosome strain with strains with fewer chromosomes, we noted two trends. As the number of chromosomes dropped below sixteen, spore viability decreased markedly, reaching less than 10% for twelve chromosomes. As the number of chromosomes decreased further, yeast sporulation was arrested: a cross between a sixteen-chromosome strain and an eight-chromosome strain showed greatly reduced full tetrad formation and less than 1% sporulation, from which no viable spores could be recovered. However, homotypic crosses between pairs of strains with eight, four or two chromosomes produced excellent sporulation and spore viability. These results indicate that eight chromosome-chromosome fusion events suffice to isolate strains reproductively. Overall, budding yeast tolerates a reduction in chromosome number unexpectedly well, providing a striking example of the robustness of genomes to change.

Inborn Errors of RNA Lariat Metabolism in Humans with Brainstem Viral Infection.

Viruses that are typically benign sometimes invade the brainstem in otherwise healthy children. We report bi-allelic DBR1 mutations in unrelated patients from different ethnicities, each of whom had brainstem infection due to herpes simplex virus 1 (HSV1), influenza virus, or norovirus. DBR1 encodes the only known RNA lariat debranching enzyme. We show that DBR1 expression is ubiquitous, but strongest in the spinal cord and brainstem. We also show that all DBR1 mutant alleles are severely hypomorphic, in terms of expression and function. The fibroblasts of DBR1-mutated patients contain higher RNA lariat levels than control cells, this difference becoming even more marked during HSV1 infection. Finally, we show that the patients' fibroblasts are highly susceptible to HSV1. RNA lariat accumulation and viral susceptibility are rescued by wild-type DBR1. Autosomal recessive, partial DBR1 deficiency underlies viral infection of the brainstem in humans through the disruption of tissue-specific and cell-intrinsic immunity to viruses.

BioPartsBuilder: a synthetic biology tool for combinatorial assembly of biological parts.

UNLABELLED: Combinatorial assembly of DNA elements is an efficient method for building large-scale synthetic pathways from standardized, reusable components. These methods are particularly useful because they enable assembly of multiple DNA fragments in one reaction, at the cost of requiring that each fragment satisfies design constraints. We developed BioPartsBuilder as a biologist-friendly web tool to design biological parts that are compatible with DNA combinatorial assembly methods, such as Golden Gate and related methods. It retrieves biological sequences, enforces compliance with assembly design standards and provides a fabrication plan for each fragment. AVAILABILITY AND IMPLEMENTATION: BioPartsBuilder is accessible at http://public.biopartsbuilder.org and an Amazon Web Services image is available from the AWS Market Place (AMI ID: ami-508acf38). Source code is released under the MIT license, and available for download at https://github.com/baderzone/biopartsbuilder CONTACT: joel.bader@jhu.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

RADOM, an efficient in vivo method for assembling designed DNA fragments up to 10 kb long in Saccharomyces cerevisiae.

We describe rapid assembly of DNA overlapping multifragments (RADOM), an improved assembly method via homologous recombination in Saccharomyces cerevisiae, which combines assembly in yeasto with blue/white screening in Escherichia coli. We show that RADOM can successfully assemble ∼3 and ∼10 kb DNA fragments that are highly similar to the yeast genome rapidly and accurately. This method was tested in the Build-A-Genome course by undergraduate students, where 125 ∼3 kb "minichunks" from the synthetic yeast genome project Sc2.0 were assembled. Here, 122 out of 125 minichunks achieved insertions with correct sizes, and 102 minichunks were sequenced verified. As this method reduces the time-consuming and labor-intensive efforts of yeast assembly by improving the screening efficiency for correct assemblies, it may find routine applications in the construction of DNA fragments, especially in hierarchical assembly projects.