Faculty Bibliography

Historians of the future may well describe 2018 as the year that the world's first functional synthetic eukaryotic genome became a reality. Without the benefit of hindsight, it might be hard to completely grasp the long-term significance of a breakthrough moment in the history of science like this. The role of synthetic biology in the imminent birth of a budding Saccharomyces cerevisiae yeast cell carrying 16 man-made chromosomes causes the world of science to teeter on the threshold of a future-defining scientific frontier. The genome-engineering tools and technologies currently being developed to produce the ultimate yeast genome will irreversibly connect the dots between our improved understanding of the fundamentals of a complex cell containing its DNA in a specialised nucleus and the application of bioengineered eukaryotes designed for advanced biomanufacturing of beneficial products. By joining up the dots between the findings and learnings from the international Synthetic Yeast Genome project (known as the Yeast 2.0 or Sc2.0 project) and concurrent advancements in biodesign tools and smart data-intensive technologies, a future world powered by a thriving bioeconomy seems realistic. This global project demonstrates how a collaborative network of dot connectors-driven by a tinkerer's indomitable curiosity to understand how things work inside a eukaryotic cell-are using cutting-edge biodesign concepts and synthetic biology tools to advance science and to positively frame human futures (i.e. improved quality of life) in a planetary context (i.e. a sustainable environment). Explorations such as this have a rich history of resulting in unexpected discoveries and unanticipated applications for the benefit of people and planet. However, we must learn from past explorations into controversial futuristic sciences and ensure that researchers at the forefront of an emerging science such as synthetic biology remain connected to all stakeholders' concerns about the biosafety, bioethics and regulatory aspects of their pioneering work. This article presents a shared vision of constructing a synthetic eukaryotic genome in a safe model organism by using novel concepts and advanced technologies. This multidisciplinary and collaborative project is conducted under a sound governance structure that does not only respect the scientific achievements and lessons from the past, but that is also focussed on leading the present and helping to secure a brighter future for all.

Quiescent cells exploit an array of transcription factors to activate stress response machinery and maintain survival under nutrient-limited conditions. Our recent findings reveal that these transcription factors also play an important role in the exit of quiescence and regrowth. By studying Saccharomyces cerevisiae under a continuous, nutrient-limited condition, we found that Msn2 and Msn4 function as master regulators of glycolytic genes in the quiescent-like phase. They control the timing of transition from quiescence to growth by regulating the accumulation rate of acetyl-CoA, a key metabolite that is downstream of glycolysis and drives growth. These findings suggest a model that Msn2/4 not only protect the cells from starvation but also facilitate their regrowth from quiescence. Thus, understanding the functions of stress response transcription factors in metabolic regulation will provide deeper insight into how quiescent cells manage the capacity of regrowth.

Vectors encoding selectable markers have been widely used in yeast to maintain or express exogenous DNA fragments. In the fission yeastSchizosaccharomyces pombe, several engineered markers have been reported and widely used, such asura4⁺andScLEU2fromSaccharomyces cerevisiae, which complementura4andleu1mutations, respectively. These two auxotrophic markers share no homology with theS. pombegenome; however, most others can recombine with the genome due to sequence homology shared between the genomic and plasmid-borne copies of the markers. Here, we describe a CRISPR-Cas9 toolbox that can be used to quickly introduce "designer" auxotrophic marker deletions into host strains, includingleu1-Δ0,his3-Δ0, andlys9-Δ0Together withura4-D18, this brings the total number of available designer deletion auxotrophic markers to four. The toolbox consists of a Cas9-gRNA expression vector and a donor DNA plasmid pair for each designer deletion. Using this toolbox, a set of auxotrophicS. pombestrains was constructed. Further, we reorganized essential components in the commonly used pREP series of plasmids and assembled the corresponding auxotrophic marker gene onto these plasmids. This toolbox for producing designer deletions, together with the newly developed strains and plasmids, will benefit the whole yeast community.

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.

Background/UNASSIGNED:Recombinant DNA technology is today a fundamental tool for virtually all biological research fields. Among the many techniques available for the construction of a "custom DNA" molecule, the isothermal in vitro assembly, or Gibson assembly, allows for an efficient, one-step, scarless recombination-based assembly. Results/UNASSIGNED:Here, we apply and characterize the use of Gibson assembly for the deletion of DNA sequences around a DNA cut. This method, that we named "Gibson Deletion", can be used to easily substitute or delete one or more restriction sites within a DNA molecule. We show that Gibson Deletion is a viable method to delete up to 100 nucleotides from the DNA ends of a cleavage site. In addition, we found that Gibson Deletion can be performed using single strand DNA with the same efficiency as using double strand DNA molecules. Conclusions/UNASSIGNED:Gibson Deletion is a novel, easy and convenient application of isothermal in vitro assembly, that performs with high efficiency and can be implemented for a broad range of applications.

Biological processes are usually associated with genome-wide remodeling of transcription driven by transcription factors (TFs). Identifying key TFs and their spatiotemporal binding patterns are indispensable to understanding how dynamic processes are programmed. However, most methods are designed to predict TF binding sites only. We present a computational method, dynamic motif occupancy analysis (DynaMO), to infer important TFs and their spatiotemporal binding activities in dynamic biological processes using chromatin profiling data from multiple biological conditions such as time-course histone modification ChIP-seq data. In the first step, DynaMO predicts TF binding sites with a random forests approach. Next and uniquely, DynaMO infers dynamic TF binding activities at predicted binding sites using their local chromatin profiles from multiple biological conditions. Another landmark of DynaMO is to identify key TFs in a dynamic process using a clustering and enrichment analysis of dynamic TF binding patterns. Application of DynaMO to the yeast ultradian cycle, mouse circadian clock and human neural differentiation exhibits its accuracy and versatility. We anticipate DynaMO will be generally useful for elucidating transcriptional programs in dynamic processes.

LINE-1/L1 retrotransposon sequences comprise 17% of the human genome. Among the many classes of mobile genetic elements, L1 is the only autonomous retrotransposon that still drives human genomic plasticity today. Through its co-evolution with the human genome, L1 has intertwined itself with host cell biology. However, a clear understanding of L1's lifecycle and the processes involved in restricting its insertion and intragenomic spread remains elusive. Here we identify modes of L1 proteins' entrance into the nucleus, a necessary step for L1 proliferation. Using functional, biochemical, and imaging approaches, we also show a clear cell cycle bias for L1 retrotransposition that peaks during the S phase. Our observations provide a basis for novel interpretations about the nature of nuclear and cytoplasmic L1 ribonucleoproteins (RNPs) and the potential role of DNA replication in L1 retrotransposition.

Long Interspersed Nuclear Element-1 (LINE-1, L1) is a mobile genetic element active in human genomes. L1-encoded ORF1 and ORF2 proteins bind L1 RNAs, forming ribonucleoproteins (RNPs). These RNPs interact with diverse host proteins, some repressive and others required for the L1 lifecycle. Using differential affinity purifications, quantitative mass spectrometry, and next generation RNA sequencing, we have characterized the proteins and nucleic acids associated with distinctive, enzymatically active L1 macromolecular complexes. Among them, we describe a cytoplasmic intermediate that we hypothesize to be the canonical ORF1p/ORF2p/L1-RNA-containing RNP, and we describe a nuclear population containing ORF2p, but lacking ORF1p, which likely contains host factors participating in target-primed reverse transcription.

The ability to express non-native pathways in genetically tractable model systems is important for fields such as synthetic biology, genetics, and metabolic engineering. Here we describe a modular and hierarchical strategy to assemble multigene pathways for expression in S. cerevisiae. First, discrete promoter, coding sequence, and terminator parts are assembled in vitro into Transcription Units (TUs) flanked by adapter sequences using "yeast Golden Gate" (yGG), a type IIS restriction enzyme-dependent cloning strategy. Next, harnessing the natural capacity of S. cerevisiae for homologous recombination, TUs are assembled into pathways and expressed using the "Versatile Genetic Assembly System" (VEGAS) in yeast. Coupling transcription units constructed by yGG with VEGAS assembly is a generic and flexible workflow to achieve pathway expression in S. cerevisiae. This protocol describes assembly of a five TU pathway for yeast production of violacein, a pigment derived from Chromobacterium violaceum.

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 (DSB) at the MAT locus, and in a single co-transformation, both haploid and diploid cells were switched to the specified mating-type at ~80% efficiency. The mating-type of strains carrying either rod or ring chromosome III were switched, including those lacking HMLalpha and HMRa cryptic mating loci. Furthermore, we transplanted the synthetic yeast chromosome V to build a haploid poly-synthetic 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/a/a isogenic tetraploids.