Standard methods for transgenesis in zebrafish depend on random transgene integration into the genome followed by resource-intensive screening and validation. Targeted vector integration into validated genomic loci using phiC31 integrase-based attP/attB recombination has transformed mouse and Drosophila transgenesis. However, while the phiC31 system functions in zebrafish, validated loci carrying attP-based landing or safe harbor sites for transgene integration in zebrafish have not been established. Here, using CRISPR-Cas9, we converted two well-validated single insertion Tol2-based zebrafish transgenes with long-standing genetic stability into two attP landing sites, called phiC31 Integrase Genomic Loci Engineered for Transgenesis (pIGLET). Generating fluorescent reporters, loxP-based Switch lines, CreERT2 effector transgenes, and gene-regulatory variant reporters in the pIGLET14a and pIGLET24b landing site alleles, we document their suitability for transgenesis applications across cell types and developmental stages. For both landing sites, we routinely achieve 25-50% germline transmission of targeted transgene integrations, drastically reducing the number of required animals and necessary resources to generate individual transgenic lines. We document that phiC31 integrase-based transgenesis into pIGLET14a and pIGLET24b reproducibly results in representative reporter expression patterns in injected F0 zebrafish embryos for enhancer discovery and enables qualitative and quantitative comparison of gene-regulatory element variants. Taken together, our new phiC31 integrase-based transgene landing sites establish reproducible, targeted zebrafish transgenesis for numerous applications including quantitative experiments while greatly reducing the workload of generating new transgenic zebrafish lines. less

Expression quantitative trait loci (eQTLs) provide a key bridge between noncoding DNA sequence variants and organismal traits. The effects of eQTLs can differ among tissues, cell types, and cellular states, but these differences are obscured by gene expression measurements in bulk populations. We developed a one-pot approach to map eQTLs in Saccharomyces cerevisiae by single-cell RNA sequencing (scRNA-seq) and applied it to over 100,000 single cells from three crosses. We used scRNA-seq data to genotype each cell, measure gene expression, and classify the cells by cell-cycle stage. We mapped thousands of local and distant eQTLs and identified interactions between eQTL effects and cell-cycle stages. We took advantage of single-cell expression information to identify hundreds of genes with allele-specific effects on expression noise. We used cell-cycle stage classification to map 20 loci that influence cell-cycle progression. One of these loci influenced the expression of genes involved in the mating response. We showed that the effects of this locus arise from a common variant (W82R) in the gene GPA1, which encodes a signaling protein that negatively regulates the mating pathway. The 82R allele increases mating efficiency at the cost of slower cell-cycle progression and is associated with a higher rate of outcrossing in nature. Our results provide a more granular picture of the effects of genetic variants on gene expression and downstream traits. less

Gene drive technology has the potential to address major biological challenges, including the management of disease vectors, invasive species, and agricultural pests. After releasing individuals carrying the gene drive in the target population, suppression gene drives are designed to spread at a rapid rate and carry a recessive fitness cost, thus bringing about a decline in population size or even complete suppression. Well-studied homing suppression drives have been shown to be highly efficient in Anopheles mosquitoes and were successful in eliminating large cage populations. However, for other organisms, including Aedes mosquitoes, homing gene drives are so far too inefficient to achieve complete population suppression, mainly due to lower rates of drive conversion, which is the rate at which wild type alleles are converted into drive alleles. Low drive conversion is also a major issue in vertebrates, as indicated by experiments in mice. To tackle this issue, we propose a novel gene drive design that has two targets: a homing site where the drive is located and drive conversion takes place (with rescue for an essential gene), and a distant site for providing the fitness cost for population suppression (preferably a female fertility gene, for which no rescue is provided). We modeled this design and found that the two-target system allows suppression to occur over a much wider range of drive conversion efficiency. Specifically, in the new design, the suppressive power depends mostly on total gRNA cutting efficiency instead of just drive conversion efficiency, which is advantageous because cut rates are often substantially higher than drive conversion rates. We constructed a proof of concept in Drosophila melanogaster and show that both components of the gene drive function successfully. However, embryo drive activity from maternally deposited Cas9 as well as fitness costs for female drive heterozygotes both remain significant challenges for two-target and standard suppression drives. Overall, our improved gene drive design eases the development of strong homing suppression gene drives for many species where drive conversion is less efficient. less

Human craniofacial shape is highly variable yet highly heritable with genetic variants interacting through multiple layers of development. Here, we hypothesize that Mendelian phenotypes represent the extremes of a phenotypic spectrum and, using achondroplasia as an example, we introduce a syndrome-informed phenotyping approach to identify genomic loci associated with achondroplasia-like facial variation in the normal population. We compared three-dimensional facial scans from 43 individuals with achondroplasia and 8246 controls to calculate achondroplasia-like facial scores. Multivariate GWAS of the control scores revealed a polygenic basis for normal facial variation along an achondroplasia-specific shape axis, identifying genes primarily involved in skeletal development. Jointly modeling these genes in two independent control samples showed craniofacial effects approximating the characteristic achondroplasia phenotype. These findings suggest that both complex and Mendelian genetic variation act on the same developmentally determined axes of facial variation, providing new insights into the genetic intersection of complex traits and Mendelian disorders. less

Myocyte Enhancer Factor 2C (MEF2C) is a transcription factor that plays a crucial role in neurogenesis and synapse development. Genetic studies have identified MEF2C as a gene that influences cognition and risk for neuropsychiatric disorders, including autism spectrum disorder (ASD) and schizophrenia (SCZ). Here, we investigated the involvement of MEF2C in these phenotypes using human-derived neural stem cells (NSCs) and induced neurons (iNs), which represented early and late neurodevelopmental stages. For these cellular models, MEF2C function had previously been disrupted, either by direct or indirect mutation, and gene expression assayed using RNA-seq. We integrated these RNA-seq data with MEF2C ChIP-seq data to identify dysregulated direct target genes of MEF2C in the NSCs and iNs models. Several MEF2C direct target gene-sets were enriched for SNP-based heritability for intelligence, educational attainment and SCZ, as well as being enriched for genes containing rare de novo mutations reported in ASD and/or developmental disorders. These gene-sets are enriched in both excitatory and inhibitory neurons in the hippocampus and cortex and are involved in a wide range of biological processes including neurogenesis, metabolic processes, protein modifications, as well as mitochondrial function and energy production. We observed a trans expression quantitative trait locus (eQTL) effect of a single SNP at MEF2C (rs6893807, which is associated with IQ) on the expression of a target gene, BNIP3L. BNIP3L is a prioritized risk gene from the largest genome-wide association study of SCZ and has a function in mitophagy in mitochondria. Overall, our analysis reveals that either direct or indirect disruption of MEF2C dysregulates sets of genes that contain multiple alleles associated with SCZ risk and cognitive function and implicates neurogenesis and mitochondrial function in the etiology of these phenotypes. less

SIRT5 is a sirtuin deacylase that represents the major activity responsible for removal of negatively-charged lysine modifications, in the mitochondrial matrix and elsewhere in the cell. In benign cells and mouse models, under basal non-stressed conditions, the phenotypes of SIRT5 deficiency are generally quite subtle. Here, we identify two homozygous SIRT5 variants in human patients suffering from severe mitochondrial disease. Both variants, P114T and L128V, are associated with reduced SIRT5 protein stability and impaired biochemical activity, with no evidence of neomorphic or dominant negative properties. The crystal structure of the P114T enzyme was solved and shows only subtle deviations from wild-type. Via CRISPR-Cas9, we generate a mouse model that recapitulates the human P114T mutation; homozygotes show reduced SIRT5 levels and activity, but no obvious metabolic abnormalities, neuropathology or other gross evidence of severe disease. We conclude that these human SIRT5 variants most likely represent severe hypomorphs, and are likely not the primary pathogenic cause of the neuropathology observed in the patients. less

Fusarium diseases cause serious damage to agricultural crops. In agricultural practice, various chemical fungicides, usually based on triazole, are often used to control fusariosis. This leads to the emergence of resistant forms among pathogenic fungi. An alternative to chemical fungicides treatments may be the use of biopreparations based on natural antagonists of Fusarium. Bacillus and Paenibacillus bacteria isolated from farmland soils exhibit a high level of antagonism against phytopathogenic fungi of the genus Fusarium. PCR-based analysis revealed that genes of non-ribosomal peptide synthases are present in the genomes of isolated bacterial strains, while these strains do not have any chitinase activity, so the antifungal effect seems to be provided predominantly by non-ribosomally synthesized peptides. The expression of the detected genes is increased in the presence of fungi during co-culturing, and this effect is strain-specific and pathogen-dependent. In light of these findings, the development of pathogen-specific drugs seems promising. less

In high dimensional observational genotype-phenotype data, complex relationships and confounders make causal learning difficult. Here, we bridge a gap between genetic epidemiology and statistical causal inference, to demonstrate that graphical inference can fine-map trait-specific causal DNA variants and identify risk factors that are most likely to have a direct causal effect on a disease outcome. Our CI-GWAS approach learns a single graph representing the causal relationships among millions of DNA variants and 17 traits in less than 10 minutes on standard GPU architecture. We find over 100 causal trait-specific DNA variants that are exclusively exonic, with clear pathways from trait-specific core genes to each outcome. We separate pleiotropy from linkage to find evidence that PCSK9, LPA, and RP1-81D8.3 are pleiotropic for cardiovascular disease (CAD) with blood cholesterol, triglycerides, and low-density lipoprotiens respectively. CI-GWAS accounts for pleiotropy and selects waist-hip ratio, alcohol consumption and smoking as directly causal for CAD, with many other variables having complex paths linked through these risk factors. Our work facilitates extensive investigation of potential causal hypotheses across a wide-range of data. less

Currently, there exists conflicting data regarding the biological activity of unmodified fullerene C60. Various sources report its toxicity, geroprotective activity, and potential interaction with DNA. Contradictory findings regarding the toxicity of C60 may arise from the use of toxic solvents, as well as the influence of bioavailability and bioactivity on the preparation conditions of C60 suspensions. Furthermore, the microbiota of experimental animals can impact geroprotective activity results by releasing surfactants that facilitate substance penetration through the cell membrane. In this study, we selected conditions for solubilizing fullerene C60 in a solution of surfactin, a surfactant of bacterial origin, as well as in a 2% aqueous solution of TWEEN 80, employing ultrasound. Through bioluminescent analysis using lux biosensors in E. coli MG1655, we observed that C60 in surfactin reduced induced genotoxic and oxidative stress. Given that surfactin enhances membrane permeability to fullerene C60, suspensions of fullerene in designated concentrations of surfactin can be regarded as a DNA protector and antioxidant, warranting further investigation as a promising component of novel drugs. less

Comparative approaches have revealed both divergent and convergent paths to achieving shared developmental outcomes. Thus, only through assembling multiple case studies can we understand biological principles. Yet, despite appreciating the conservation or lack thereof of developmental networks, the conservation of epigenetic mechanisms regulating these networks is poorly understood. The nematode Pristionchus pacificus has emerged as a model system of plasticity and epigenetic regulation as it exhibits a bacterivorous or omnivorous morph depending on its environment. Here, we determined the epigenetic toolkit available to P. pacificus as a resource for future functional work on plasticity, and as a comparison with C. elegans to investigate the conservation of epigenetic mechanisms. Broadly, we observed a similar cast of genes with putative epigenetic function between C. elegans and P. pacificus. However, we also found striking differences. Most notably, the histone methyltransferase complex PRC2 appears to be missing in P. pacificus.We described the deletion/pseudogenization of the PRC2 genes mes-2 and mes-6 and concluded that both were lost in the last common ancestor of P. pacificus and a related species P. arcanus. Interestingly, we observed the enzymatic product of PRC2 (H3K27me3) by mass spectrometry and immunofluorescence, suggesting that a currently unknown methyltransferase has been co-opted for heterochromatin silencing. Altogether, we have provided an inventory of epigenetic genes in P. pacificus to enable reverse-genetic experiments related to plasticity, and in doing so have described the first loss of PRC2 in a multicellular organism. less