Germ cells do not progress through spermatogenesis in the infertile zebrafish testis.

Vertebrate spermatogonial stem cells maintain sperm production over the lifetime of an animal but fertility declines with age. While morphological studies have greatly informed our understanding of typical spermatogenesis, the molecular and cellular mechanisms underlying spermatogenesis are not yet understood, particularly with respect to the onset of fertility. We used single-cell RNA sequencing to generate a developmental atlas of the zebrafish testis. Using 5 timepoints across the adult life of a zebrafish, we described cellular profiles in the testis during and after fertility. While all germ cell stages of spermatogenesis are detected in testes from fertile adult zebrafish, testes from older infertile males only contained spermatogonia and a reduced population of spermatocytes. These remaining germ cells are transcriptionally distinct from fertile spermatogonia. Immune cells including macrophages and lymphocytes drastically increase in abundance in infertile testes. Our developmental atlas reveals the cellular changes as the testis ages and defines a molecular roadmap for the regulation of male fertility.

Characterization of Bud3 domains sufficient for bud neck targeting in S. cerevisiae.

The cytoskeleton serves a diverse set of functions in both multi- and unicellular organisms, including movement, transport, morphology, cell division and cell signalling. The septin family of cytoskeletal proteins are found within all fungi and metazoans and can generate three-dimensional scaffolds in vivo that promote membrane curvature, serve as physical barriers and coordinate cell cycle checkpoints. In budding yeast, the septins organize into polymerized filaments that decorate the division site between mother and daughter cells during mitosis; assembly of this structure at the 'bud neck' is critical for completion of cytokinesis and execution of numerous other cellular events. One such pathway includes bud site selection and the recruitment of proteins such as Bud4 and Bud3 that are responsible for promoting an axial budding pattern in haploid yeast. While Bud4 appears to be recruited to the septins independently of the presence of Bud3, it is likely that Bud3 can localize to the bud neck using both Bud4-dependent and Bud4-independent mechanisms. Furthermore, it remains unclear which precise domain or domains within Bud3 is/are both necessary and sufficient for optimal association at the septin structure. In this study, we examined the localization of GFP-Bud3 constructs in otherwise wild-type (WT) haploid yeast cells expressing Cdc10-mCherry using fluorescence microscopy; we tested a collection of N- and C-terminal truncations and fusions of separate Bud3 protein elements to identify the smallest domain(s) responsible for bud neck localization. We found that the coordinate action of the central amphipathic helix (residues 847-865) and a partially conserved C-terminal motif (residues 1172-1273) was sufficient to promote bud neck recruitment in the presence of endogenous Bud3. This domain is considerably smaller than the previously characterized C-terminal portion required to physically interact with Bud4 (1221-1636) and utilizes a similar mechanism of pairing membrane association, with a separate localization domain, similar to other non-septin proteins targeted to the division site during cell division.

Gene drive inhibition by the anti-CRISPR proteins AcrIIA2 and AcrIIA4 in Saccharomyces cerevisiae.

Given the widespread use and application of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas gene editing system across many fields, a major focus has been the development, engineering and discovery of molecular means to precisely control and regulate the enzymatic function of the Cas9 nuclease. To date, a variety of Cas9 variants and fusion assemblies have been proposed to provide temporally inducible and spatially controlled editing functions. The discovery of a new class of 'anti-CRISPR' proteins, evolved from bacteriophage in response to the prokaryotic nuclease-based immune system, provides a new platform for control over genomic editing. One Cas9-based application of interest to the field of population control is that of the 'gene drive'. Here, we demonstrate use of the AcrIIA2 and AcrIIA4 proteins to inhibit active gene drive systems in budding yeast. Furthermore, an unbiased mutational scan reveals that titration of Cas9 inhibition may be possible by modification of the anti-CRISPR primary sequence.

Tuning CRISPR-Cas9 Gene Drives in Saccharomyces cerevisiae.

Control of biological populations is an ongoing challenge in many fields, including agriculture, biodiversity, ecological preservation, pest control, and the spread of disease. In some cases, such as insects that harbor human pathogens (e.g., malaria), elimination or reduction of a small number of species would have a dramatic impact across the globe. Given the recent discovery and development of the CRISPR-Cas9 gene editing technology, a unique arrangement of this system, a nuclease-based "gene drive," allows for the super-Mendelian spread and forced propagation of a genetic element through a population. Recent studies have demonstrated the ability of a gene drive to rapidly spread within and nearly eliminate insect populations in a laboratory setting. While there are still ongoing technical challenges to design of a more optimal gene drive to be used in wild populations, there are still serious ecological and ethical concerns surrounding the nature of this powerful biological agent. Here, we use budding yeast as a safe and fully contained model system to explore mechanisms that might allow for programmed regulation of gene drive activity. We describe four conserved features of all CRISPR-based drives and demonstrate the ability of each drive component-Cas9 protein level, sgRNA identity, Cas9 nucleocytoplasmic shuttling, and novel Cas9-Cas9 tandem fusions-to modulate drive activity within a population.

CRISPR-UnLOCK: Multipurpose Cas9-Based Strategies for Conversion of Yeast Libraries and Strains.

Saccharomyces cerevisiae continues to serve as a powerful model system for both basic biological research and industrial application. The development of genome-wide collections of individually manipulated strains (libraries) has allowed for high-throughput genetic screens and an emerging global view of this single-celled Eukaryote. The success of strain construction has relied on the innate ability of budding yeast to accept foreign DNA and perform homologous recombination, allowing for efficient plasmid construction (in vivo) and integration of desired sequences into the genome. The development of molecular toolkits and "integration cassettes" have provided fungal systems with a collection of strategies for tagging, deleting, or over-expressing target genes; typically, these consist of a C-terminal tag (epitope or fluorescent protein), a universal terminator sequence, and a selectable marker cassette to allow for convenient screening. However, there are logistical and technical obstacles to using these traditional genetic modules for complex strain construction (manipulation of many genomic targets in a single cell) or for the generation of entire genome-wide libraries. The recent introduction of the CRISPR/Cas gene editing technology has provided a powerful methodology for multiplexed editing in many biological systems including yeast. We have developed four distinct uses of the CRISPR biotechnology to generate yeast strains that utilizes the conversion of existing, commonly-used yeast libraries or strains. We present Cas9-based, marker-less methodologies for (i) N-terminal tagging, (ii) C-terminally tagging yeast genes with 18 unique fusions, (iii) conversion of fluorescently-tagged strains into newly engineered (or codon optimized) variants, and finally, (iv) use of a Cas9 "gene drive" system to rapidly achieve a homozygous state for a hypomorphic query allele in a diploid strain. These CRISPR-based methods demonstrate use of targeting universal sequences previously introduced into a genome.