The CRISPR-Cas9 system is extremely useful for genome editing in many species, including the model yeast Saccharomyces cerevisiae, and other yeast species. We have previously reported the use of an inducible CRISPR-Cas9 system in Candida glabrata, which allows genome editing but also the study of Double Strand Break (DSB) repair. We report, in this study, a comparable system for C. glabrata, relying on a new plasmid, which is more stable than the previous one. We also report the use of this plasmid to induce DSBs in two additional human pathogens, Candida bracarensis and Candida nivariensis. We examine lethality induced by an in vivo DSB in the three species and describe the different types of Non-Homologous End-Joining (NHEJ) events detected in these three pathogens.

Yeasts in the subphylum Saccharomycotina are found across the globe in disparate ecosystems. A major aim of yeast research is to understand the diversity and evolution of ecological traits, such as carbon metabolic breadth, insect association, and cactophily. This includes studying aspects of ecological traits like genetic architecture or association with other phenotypic traits. Genomic resources in the Saccharomycotina have grown rapidly. Ecological data, however, are still limited for many species, especially those only known from species descriptions where usually only a limited number of strains are studied. Moreover, ecological information is recorded in natural language format limiting high throughput computational analysis. To address these limitations, we developed an ontological framework for the analysis of yeast ecology. A total of 1,088 yeast strains were added to the Ontology of Yeast Environments (OYE) and analyzed in a machine-learning framework to connect genotype to ecology. This framework is flexible and can be extended to additional isolates, species, or environmental sequencing data. Widespread adoption of OYE would greatly aid the study of macroecology in the Saccharomycotina subphylum.

Genomes from yeast to human are subject to pervasive transcription. A single round of pervasive transcription is sufficient to alter local chromatin conformation, nucleosome dynamics and gene expression, but experimentally is can be hard to distinguish from background signals. Size selected native elongating transcript sequencing (ssNET-Seq) was developed to precisely map transcription units (TU) independent of expression levels. RNAPII-associated nascent transcripts are fractionation into different size ranges before library construction. When anchored to the transcription start sites (TSS) of annotated genes, the combined pattern of the output metagenes define the expected reference pattern for a TU. Bioinformatic pattern matching to the reference identified 9542 TU in Saccharomyces cerevisiae, of which 47% are coding and 53% are non-coding. 3113 (33%) are newly identified unannotated non-coding TU. Anchoring all TU to the TSS or polyadenylation site (PAS) of annotated coding regions reveals distinctive architectures of linked pairs of divergent TU approximately 200nt apart. The Reb1 transcription factor is enriched 30nt downstream of the PAS only when an upstream (TSS-60nt) non-coding TU co-occurs with a downstream (TSS+150nt) coding TU and supports nucleosome depletion in the generation of the pervasive nascent transcriptome. The potential for extensive transcriptional interference is evident from low abundance unannotated TUs with variable TSS (median-240nt) initiating within a 500nt window upstream of, and transcribing over, the promoters of protein coding genes. This study confirms a highly interleaved yeast genome with different types of transcription units altering the chromatin landscape in distinctive ways, with the potential to exert extensive regulatory control.

Polyadenylation occurs at numerous sites within 3’ untranslated regions (3’ UTRs) but rarely within coding regions. How does Pol II travel through long coding regions without generating poly(A) sites, yet then permits promiscuous polyadenylation once it reaches the 3’ UTR? The cleavage/polyadenylation (CpA) machinery preferentially associates with 3’ UTRs, but it is unknown how its recruitment is restricted to 3’ UTRs during Pol II elongation. Unlike coding regions, 3’ UTRs have long AT-rich stretches of DNA that may be important for restricting polyadenylation to 3’ UTRs. Recognition of the 3’ UTR could occur at the DNA (AT-rich), RNA (AU-rich), or RNA:DNA hybrid rU:dA- and/or rA:dT-rich) level. Based on the nucleic acid critical for 3’ UTR recognition, there are three classes of models, not mutually exclusive, for how the CpA machinery is selectively recruited to 3’ UTRs, thereby restricting where polyadenylation occurs: 1) RNA-based models suggest that the CpA complex directly (or indirectly through one or more intermediary proteins) binds long AU-rich stretches that are exposed after Pol II passes through these regions. 2) DNA-based models suggest that the AT-rich sequence affects nucleosome depletion or the elongating Pol II machinery, resulting in dissociation of some elongation factors and subsequent recruitment of the CpA machinery. 3) RNA:DNA hybrid models suggest that preferential destabilization of the Pol II elongation complex at rU:dA- and/or rA:dT-rich duplexes bridging the nucleotide addition and RNA exit sites permits preferential association of the CpA machinery with 3’ UTRs. Experiments to provide evidence for one or more of these models are suggested.

Yeast research is entering into a new period of scholarship, with new scientific tools, new questions to ask, and new issues to consider. The politics of emerging and critical technology can no longer be separated from the pursuit of basic science in fields, such as synthetic biology and engineering biology. Given the intensifying race for technological leadership, yeast research is likely to attract significant investment from government, and that it offers huge opportunities to the curious minded from a basic research standpoint. This article provides an overview of new directions in yeast research, and places these trends in their geopolitical context. At the highest level, yeast research is situated within the ongoing convergence of the life sciences with the information sciences. This convergent effect is most strongly pronounced in areas of AI-enabled tools for the life sciences, and the creation of synthetic genomes, minimal genomes, pan-genomes, neochromosomes and metagenomes using computer assisted design tools and methodologies. Synthetic yeast futures encompass basic and applied science questions that will be of intense interest to government and non-government funding sources. It is essential for the yeast research community to map and understand the context of their research in order to ensure their collaborations turn global challenges into research opportunities.