Meta-analysis of dispensable essential genes and their interactions with bypass suppressors.

Genes have been historically classified as essential or non-essential based on their requirement for viability. However, genomic mutations can sometimes bypass the requirement for an essential gene, challenging the binary classification of gene essentiality. Such dispensable essential genes represent a valuable model for understanding the incomplete penetrance of loss-of-function mutations often observed in natural populations. Here, we compiled data from multiple studies on essential gene dispensability in Saccharomyces cerevisiae to comprehensively characterize these genes. In analyses spanning different evolutionary timescales, dispensable essential genes exhibited distinct phylogenetic properties compared with other essential and non-essential genes. Integration of interactions with suppressor genes that can bypass the gene essentiality revealed the high functional modularity of the bypass suppression network. Furthermore, dispensable essential and bypass suppressor gene pairs reflected simultaneous changes in the mutational landscape of S. cerevisiae strains. Importantly, species in which dispensable essential genes were non-essential tended to carry bypass suppressor mutations in their genomes. Overall, our study offers a comprehensive view of dispensable essential genes and illustrates how their interactions with bypass suppressors reflect evolutionary outcomes.

Global analysis of suppressor mutations that rescue human genetic defects.

BACKGROUND: Genetic suppression occurs when the deleterious effects of a primary "query" mutation, such as a disease-causing mutation, are rescued by a suppressor mutation elsewhere in the genome. METHODS: To capture existing knowledge on suppression relationships between human genes, we examined 2,400 published papers for potential interactions identified through either genetic modification of cultured human cells or through association studies in patients. RESULTS: The resulting network encompassed 476 unique suppression interactions covering a wide spectrum of diseases and biological functions. The interactions frequently linked genes that operate in the same biological process. Suppressors were strongly enriched for genes with a role in stress response or signaling, suggesting that deleterious mutations can often be buffered by modulating signaling cascades or immune responses. Suppressor mutations tended to be deleterious when they occurred in absence of the query mutation, in apparent contrast with their protective role in the presence of the query. We formulated and quantified mechanisms of genetic suppression that could explain 71% of interactions and provided mechanistic insight into disease pathology. Finally, we used these observations to predict suppressor genes in the human genome. CONCLUSIONS: The global suppression network allowed us to define principles of genetic suppression that were conserved across diseases, model systems, and species. The emerging frequency of suppression interactions among human genes and range of underlying mechanisms, together with the prevalence of suppression in model organisms, suggest that compensatory mutations may exist for most genetic diseases.

Exploring conditional gene essentiality through systems genetics approaches in yeast.

An essential gene encodes for a cellular function that is required for viability. Although viability is a straightforward phenotype to analyze in yeast, defining a gene as essential is not always trivial. Gene essentiality has generally been studied in specific laboratory strains and under standard growth conditions, however, essentiality can vary across species, strains, and environments. Recent systematic studies of gene essentiality revealed that two sets of essential genes exist: core essential genes that are always required for viability and conditional essential genes that vary in essentiality in different genetic and environmental contexts. Here, we review recent advances made in the systematic analysis of gene essentiality in yeast and discuss the properties that distinguish core from context-dependent essential genes.

Predicting base editing outcomes using position-specific sequence determinants.

CRISPR/Cas base editors promise nucleotide-level control over DNA sequences, but the determinants of their activity remain incompletely understood. We measured base editing frequencies in two human cell lines for two cytosine and two adenine base editors at ∼14 000 target sequences and find that base editing activity is sequence-biased, with largest effects from nucleotides flanking the target base. Whether a base is edited depends strongly on the combination of its position in the target and the preceding base, acting to widen or narrow the effective editing window. The impact of features on editing rate depends on the position, with sequence bias efficacy mainly influencing bases away from the center of the window. We use these observations to train a machine learning model to predict editing activity per position, with accuracy ranging from 0.49 to 0.72 between editors, and with better generalization across datasets than existing tools. We demonstrate the usefulness of our model by predicting the efficacy of disease mutation correcting guides, and find that most of them suffer from more unwanted editing than pure outcomes. This work unravels the position-specificity of base editing biases and allows more efficient planning of editing campaigns in experimental and therapeutic contexts.

Chl1 helicase controls replication fork progression by regulating dNTP pools.

Eukaryotic cells have evolved a replication stress response that helps to overcome stalled/collapsed replication forks and ensure proper DNA replication. The replication checkpoint protein Mrc1 plays important roles in these processes, although its functional interactions are not fully understood. Here, we show that MRC1 negatively interacts with CHL1, which encodes the helicase protein Chl1, suggesting distinct roles for these factors during the replication stress response. Indeed, whereas Mrc1 is known to facilitate the restart of stalled replication forks, we uncovered that Chl1 controls replication fork rate under replication stress conditions. Chl1 loss leads to increased RNR1 gene expression and dNTP levels at the onset of S phase likely without activating the DNA damage response. This in turn impairs the formation of RPA-coated ssDNA and subsequent checkpoint activation. Thus, the Chl1 helicase affects RPA-dependent checkpoint activation in response to replication fork arrest by ensuring proper intracellular dNTP levels, thereby controlling replication fork progression under replication stress conditions.

Natural variants suppress mutations in hundreds of essential genes.

The consequence of a mutation can be influenced by the context in which it operates. For example, loss of gene function may be tolerated in one genetic background, and lethal in another. The extent to which mutant phenotypes are malleable, the architecture of modifiers and the identities of causal genes remain largely unknown. Here, we measure the fitness effects of ~ 1,100 temperature-sensitive alleles of yeast essential genes in the context of variation from ten different natural genetic backgrounds and map the modifiers for 19 combinations. Altogether, fitness defects for 149 of the 580 tested genes (26%) could be suppressed by genetic variation in at least one yeast strain. Suppression was generally driven by gain-of-function of a single, strong modifier gene, and involved both genes encoding complex or pathway partners suppressing specific temperature-sensitive alleles, as well as general modifiers altering the effect of many alleles. The emerging frequency of suppression and range of possible mechanisms suggest that a substantial fraction of monogenic diseases could be managed by modulating other gene products.

Systematic analysis of bypass suppression of essential genes.

Essential genes tend to be highly conserved across eukaryotes, but, in some cases, their critical roles can be bypassed through genetic rewiring. From a systematic analysis of 728 different essential yeast genes, we discovered that 124 (17%) were dispensable essential genes. Through whole-genome sequencing and detailed genetic analysis, we investigated the genetic interactions and genome alterations underlying bypass suppression. Dispensable essential genes often had paralogs, were enriched for genes encoding membrane-associated proteins, and were depleted for members of protein complexes. Functionally related genes frequently drove the bypass suppression interactions. These gene properties were predictive of essential gene dispensability and of specific suppressors among hundreds of genes on aneuploid chromosomes. Our findings identify yeast's core essential gene set and reveal that the properties of dispensable essential genes are conserved from yeast to human cells, correlating with human genes that display cell line-specific essentiality in the Cancer Dependency Map (DepMap) project.

Exploring whole-genome duplicate gene retention with complex genetic interaction analysis.

Whole-genome duplication has played a central role in the genome evolution of many organisms, including the human genome. Most duplicated genes are eliminated, and factors that influence the retention of persisting duplicates remain poorly understood. We describe a systematic complex genetic interaction analysis with yeast paralogs derived from the whole-genome duplication event. Mapping of digenic interactions for a deletion mutant of each paralog, and of trigenic interactions for the double mutant, provides insight into their roles and a quantitative measure of their functional redundancy. Trigenic interaction analysis distinguishes two classes of paralogs: a more functionally divergent subset and another that retained more functional overlap. Gene feature analysis and modeling suggest that evolutionary trajectories of duplicated genes are dictated by combined functional and structural entanglement factors.

Global Genetic Networks and the Genotype-to-Phenotype Relationship.

Genetic interactions identify combinations of genetic variants that impinge on phenotype. With whole-genome sequence information available for thousands of individuals within a species, a major outstanding issue concerns the interpretation of allelic combinations of genes underlying inherited traits. In this Review, we discuss how large-scale analyses in model systems have illuminated the general principles and phenotypic impact of genetic interactions. We focus on studies in budding yeast, including the mapping of a global genetic network. We emphasize how information gained from work in yeast translates to other systems, and how a global genetic network not only annotates gene function but also provides new insights into the genotype-to-phenotype relationship.

Genetic Network Complexity Shapes Background-Dependent Phenotypic Expression.

The phenotypic consequences of a given mutation can vary across individuals. This so-called background effect is widely observed, from mutant fitness of loss-of-function variants in model organisms to variable disease penetrance and expressivity in humans; however, the underlying genetic basis often remains unclear. Taking insights gained from recent large-scale surveys of genetic interaction and suppression analyses in yeast, we propose that the genetic network context for a given mutation may shape its propensity of exhibiting background-dependent phenotypes. We argue that further efforts in systematically mapping the genetic interaction networks beyond yeast will provide not only key insights into the functional properties of genes, but also a better understanding of the background effects and the (un)predictability of traits in a broader context.

Systematic analysis of complex genetic interactions.

To systematically explore complex genetic interactions, we constructed ~200,000 yeast triple mutants and scored negative trigenic interactions. We selected double-mutant query genes across a broad spectrum of biological processes, spanning a range of quantitative features of the global digenic interaction network and tested for a genetic interaction with a third mutation. Trigenic interactions often occurred among functionally related genes, and essential genes were hubs on the trigenic network. Despite their functional enrichment, trigenic interactions tended to link genes in distant bioprocesses and displayed a weaker magnitude than digenic interactions. We estimate that the global trigenic interaction network is ~100 times as large as the global digenic network, highlighting the potential for complex genetic interactions to affect the biology of inheritance, including the genotype-to-phenotype relationship.

Yeast Aim21/Tda2 both regulates free actin by reducing barbed end assembly and forms a complex with Cap1/Cap2 to balance actin assembly between patches and cables.

Yeast Aim21 is recruited by the SH3-containing proteins Bbc1 and Abp1 to patches and, with Tda2, reduces barbed end assembly to balance the distribution of actin between patches and cables. Aim21/Tda2 also interacts with Cap1/Cap2, revealing a complex interplay between actin assembly regulators.

Errata: Functional annotation of chemical libraries across diverse biological processes.

This corrects the article DOI: 10.1038/nchembio.2436.

Errata: Functional annotation of chemical libraries across diverse biological processes.

This corrects the article DOI: 10.1038/nchembio.2436.

Functional annotation of chemical libraries across diverse biological processes.

Chemical-genetic approaches offer the potential for unbiased functional annotation of chemical libraries. Mutations can alter the response of cells in the presence of a compound, revealing chemical-genetic interactions that can elucidate a compound's mode of action. We developed a highly parallel, unbiased yeast chemical-genetic screening system involving three key components. First, in a drug-sensitive genetic background, we constructed an optimized diagnostic mutant collection that is predictive for all major yeast biological processes. Second, we implemented a multiplexed (768-plex) barcode-sequencing protocol, enabling the assembly of thousands of chemical-genetic profiles. Finally, based on comparison of the chemical-genetic profiles with a compendium of genome-wide genetic interaction profiles, we predicted compound functionality. Applying this high-throughput approach, we screened seven different compound libraries and annotated their functional diversity. We further validated biological process predictions, prioritized a diverse set of compounds, and identified compounds that appear to have dual modes of action.

Evaluation and Design of Genome-Wide CRISPR/SpCas9 Knockout Screens.

The adaptation of CRISPR/SpCas9 technology to mammalian cell lines is transforming the study of human functional genomics. Pooled libraries of CRISPR guide RNAs (gRNAs) targeting human protein-coding genes and encoded in viral vectors have been used to systematically create gene knockouts in a variety of human cancer and immortalized cell lines, in an effort to identify whether these knockouts cause cellular fitness defects. Previous work has shown that CRISPR screens are more sensitive and specific than pooled-library shRNA screens in similar assays, but currently there exists significant variability across CRISPR library designs and experimental protocols. In this study, we reanalyze 17 genome-scale knockout screens in human cell lines from three research groups, using three different genome-scale gRNA libraries. Using the Bayesian Analysis of Gene Essentiality algorithm to identify essential genes, we refine and expand our previously defined set of human core essential genes from 360 to 684 genes. We use this expanded set of reference core essential genes, CEG2, plus empirical data from six CRISPR knockout screens to guide the design of a sequence-optimized gRNA library, the Toronto KnockOut version 3.0 (TKOv3) library. We then demonstrate the high effectiveness of the library relative to reference sets of essential and nonessential genes, as well as other screens using similar approaches. The optimized TKOv3 library, combined with the CEG2 reference set, provide an efficient, highly optimized platform for performing and assessing gene knockout screens in human cell lines.

Mechanisms of suppression: The wiring of genetic resilience.

Recent analysis of genome sequences has identified individuals that are healthy despite carrying severe disease-associated mutations. A possible explanation is that these individuals carry a second genomic perturbation that can compensate for the detrimental effects of the disease allele, a phenomenon referred to as suppression. In model organisms, suppression interactions are generally divided into two classes: genomic suppressors which are secondary mutations in the genome that bypass a mutant phenotype, and dosage suppression interactions in which overexpression of a suppressor gene rescues a mutant phenotype. Here, we describe the general properties of genomic and dosage suppression, with an emphasis on the budding yeast. We propose that suppression interactions between genetic variants are likely relevant for determining the penetrance of human traits. Consequently, an understanding of suppression mechanisms may guide the discovery of protective variants in healthy individuals that carry disease alleles, which could direct the rational design of new therapeutics.

The effect of acetaminophen on ubiquitin homeostasis in Saccharomyces cerevisiae.

Acetaminophen (APAP), although considered a safe drug, is one of the major causes of acute liver failure by overdose, and therapeutic chronic use can cause serious health problems. Although the reactive APAP metabolite N-acetyl-p-benzoquinoneimine (NAPQI) is clearly linked to liver toxicity, toxicity of APAP is also found without drug metabolism of APAP to NAPQI. To get more insight into mechanisms of APAP toxicity, a genome-wide screen in Saccharomyces cerevisiae for APAP-resistant deletion strains was performed. In this screen we identified genes related to the DNA damage response. Next, we investigated the link between genotype and APAP-induced toxicity or resistance by performing a more detailed screen with a library containing mutants of 1522 genes related to nuclear processes, like DNA repair and chromatin remodelling. We identified 233 strains that had an altered growth rate relative to wild type, of which 107 showed increased resistance to APAP and 126 showed increased sensitivity. Gene Ontology analysis identified ubiquitin homeostasis, regulation of transcription of RNA polymerase II genes, and the mitochondria-to-nucleus signalling pathway to be associated with APAP resistance, while histone exchange and modification, and vesicular transport were connected to APAP sensitivity. Indeed, we observed a link between ubiquitin levels and APAP resistance, whereby ubiquitin deficiency conferred resistance to APAP toxicity while ubiquitin overexpression resulted in sensitivity. The toxicity profile of various chemicals, APAP, and its positional isomer AMAP on a series of deletion strains with ubiquitin deficiency showed a unique resistance pattern for APAP. Furthermore, exposure to APAP increased the level of free ubiquitin and influenced the ubiquitination of proteins. Together, these results uncover a role for ubiquitin homeostasis in APAP-induced toxicity.

Mapping a diversity of genetic interactions in yeast.

Genetic interactions occur when the combination of multiple mutations yields an unexpected phenotype, and they may confound our ability to fully understand the genetic mechanisms underlying complex diseases. Genetic interactions are challenging to study because there are millions of possible different variant combinations within a given genome. Consequently, they have primarily been systematically explored in unicellular model organisms, such as yeast, with a focus on pairwise genetic interactions between loss-of-function alleles. However, there are many different types of genetic interactions, such as those occurring between gain-of-function or heterozygous mutations. Here, we review recent advances made in the systematic analysis of such diverse genetic interactions in yeast, and briefly discuss how similar studies could be undertaken in human cells.

Exploring genetic suppression interactions on a global scale.

Genetic suppression occurs when the phenotypic defects caused by a mutation in a particular gene are rescued by a mutation in a second gene. To explore the principles of genetic suppression, we examined both literature-curated and unbiased experimental data, involving systematic genetic mapping and whole-genome sequencing, to generate a large-scale suppression network among yeast genes. Most suppression pairs identified novel relationships among functionally related genes, providing new insights into the functional wiring diagram of the cell. In addition to suppressor mutations, we identified frequent secondary mutations,in a subset of genes, that likely cause a delay in the onset of stationary phase, which appears to promote their enrichment within a propagating population. These findings allow us to formulate and quantify general mechanisms of genetic suppression.