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The utilization of human cDNA libraries in yeast genetic screens is an approach that has been used to identify novel gene functions and/or genetic and physical interaction partners through forward genetics using yeast two-hybrid (Y2H) and classical cDNA library screens. Here, we summarize several challenges that have been observed during the implementation of human cDNA library screens in Saccharomyces cerevisiae (budding yeast). Upon the utilization of DNA repair deficient-yeast strains to identify novel genes that rescue the toxic effect of DNA-damage inducing drugs, we have observed a wide range of transcripts that could rescue the strains. However, after several rounds of screening, most of these hits turned out to be false positives, most likely due to spontaneous mutations in the yeast strains that arise as a rescue mechanism due to exposure to toxic DNA damage inducing-drugs.The observed transcripts included mitochondrial hits, non-coding RNAs, truncated cDNAs, and transcription products that resulted from the internal priming of genomic regions. We have also noticed that most cDNA transcripts are not fused with the GAL4 activation domain (GAL4AD), rendering them unsuitable for Y2H screening. Consequently, we utilized Sanger sequencing to screen 282 transcripts obtained from either four different yeast screens or through direct fishing from a human kidney cDNA library. The aim was to gain insights into the different transcription products and to highlight the challenges of cDNA screening approaches in the presence of a significant number of undesired transcription products. In summary, this study describes the challenges encountering human cDNA library screening in yeast as a valuable technique that led to the identification of important molecular mechanisms. The results open research venues to further optimize the process and increase its efficiency.

Advances of in vitro culture models have allowed unprecedented insights into human neurobiology. At the same time genetic screening has matured into a robust and accessible experimental strategy allowing for the simultaneous study of many genes in parallel. The combination of both technologies is a newly emerging tool for neuroscientists, opening the door to identifying causal cell- and tissue-specific developmental and disease mechanisms. However, with complex experimental genetic screening set-ups new challenges in data interpretation and experimental scope arise that require a deep understanding of the benefits and challenges of individual approaches. In this review, we summarize the literature that applies genetic screening to in vitro brain models, compare experimental strengths and weaknesses and point towards future directions of these promising approaches. [ABSTRACT FROM AUTHOR]

Functional genetic screens to uncover tumor-intrinsic nodes of immune resistance have uncovered numerous mechanisms by which tumors evade our immune system. However, due to technical limitations, tumor heterogeneity is imperfectly captured with many of these analyses. Here, we provide an overview of the nature and sources of heterogeneity that are relevant for tumor-immune interactions. We argue that this heterogeneity may actually contribute to the discovery of novel mechanisms of immune evasion, given a sufficiently large and heterogeneous set of input data. Taking advantage of tumor cell heterogeneity, we provide proof-of-concept analyses of mechanisms of TNF resistance. Thus, consideration of tumor heterogeneity is imperative to increase our understanding of immune resistance mechanisms. [ABSTRACT FROM AUTHOR]

Functional genetic screens to uncover tumor-intrinsic nodes of immune resistance have uncovered numerous mechanisms by which tumors evade our immune system. However, due to technical limitations, tumor heterogeneity is imperfectly captured with many of these analyses. Here, we provide an overview of the nature and sources of heterogeneity that are relevant for tumor-immune interactions. We argue that this heterogeneity may actually contribute to the discovery of novel mechanisms of immune evasion, given a sufficiently large and heterogeneous set of input data. Taking advantage of tumor cell heterogeneity, we provide proof-of-concept analyses of mechanisms of TNF resistance. Thus, consideration of tumor heterogeneity is imperative to increase our understanding of immune resistance mechanisms.

The brain is arguably the most complex part of the human body in form and function. Much remains unclear about the molecular mechanisms that regulate its normal and pathological physiology. This lack of knowledge largely stems from the inaccessible nature of the human brain, and the limitation of animal models. As a result, brain disorders are difficult to understand and even more difficult to treat. Recent advances in generating human pluripotent stem cells (hPSCs)-derived 2-dimensional (2D) and 3-dimensional (3D) neural cultures have provided an accessible system to model the human brain. Breakthroughs in gene editing technologies such as CRISPR/Cas9 further elevate the hPSCs into a genetically tractable experimental system. Powerful genetic screens, previously reserved for model organisms and transformed cell lines, can now be performed in human neural cells. Combined with the rapidly expanding single-cell genomics toolkit, these technological advances culminate to create an unprecedented opportunity to study the human brain using functional genomics. This review will summarize the current progress of applying CRISPR-based genetic screens in hPSCs-derived 2D neural cultures and 3D brain organoids. We will also evaluate the key technologies involved and discuss their related experimental considerations and future applications.

Oxygen‐sensing mechanisms have evolved to allow organisms to respond and adapt to oxygen availability. In metazoans, oxygen‐sensing is predominantly mediated by the hypoxia inducible factors (HIFs). These transcription factors are stabilised when oxygen is limiting, activating genes involved in angiogenesis, cell growth, pH regulation and metabolism to reset cell function and adapt to the cellular environment. However, the recognition that other cellular pathways and enzymes can also respond to changes in oxygen abundance provides further complexity. Dissecting this interplay of oxygen‐sensing mechanisms has been a key research goal. Here, we review how genetic approaches have contributed to our knowledge of oxygen‐sensing pathways which to date have been predominantly focused on the HIF pathway. We discuss how genetic studies have advanced the field and outline the implications and limitations of such approaches for the development of therapies targeting oxygen‐sensing mechanisms in human disease. [ABSTRACT FROM AUTHOR]

Drosophila has been long appreciated as a classic genetic system for its ability to define gene function in vivo. Within the last several decades, the fly has also emerged as a premiere system for modeling and defining mechanisms of human disease by expressing dominant human disease genes and analyzing the effects. Here I discuss key aspects of this latter approach that first intrigued me to focus my laboratory research on this idea. Differences between the loss-of-function vs. the gain-of-function approach are raised—and the insight of these approaches for appreciating mechanisms that contribute to human neurodegenerative disease. The application of modifier genetics, which is a prominent goal of models of human disease, has implications for how specific genes or pathways intersect with the dominant disease-associated mechanisms. Models of human disease will continue to reveal unanticipated insight into fundamental cellular processes—insight that might be harder to glean from classical genetic methodologies vs modifier genetics of disease.

Experimental embryologists working at the turn of the 19th century suggested fundamental mechanisms of development, such as localized cytoplasmic determinants and tissue induction. However, the molecular basis underlying these processes proved intractable for a long time, despite concerted efforts in many developmental systems to isolate factors with a biological role. That road block was overcome by combining developmental biology with genetics. This powerful approach used unbiased genome-wide screens to isolate mutants with developmental defects and to thereby identify genes encoding key determinants and regulatory pathways that govern development. Two small invertebrates were the pioneers: the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans. Their modes of development differ in many ways, but the two together led the way to unraveling the molecular mechanisms of many fundamental developmental processes. The discovery of the grand homologies between key players in development throughout the animal kingdom underscored the usefulness of studying these small invertebrate models for animal development and even human disease. We describe developmental genetics in Drosophila and C. elegans up to the rise of genomics at the beginning of the 21st Century. Finally, we discuss themes that emerge from the histories of such distinct organisms and prospects of this approach for the future. [Display omitted] • Genome-wide mutant screens in the fly Drosophila and the worm C.elegans made major contributions to Developmental Biology. • The screens identified genes encoding key determinants and regulatory pathways that govern development. • C. elegans and Drosophila are very different invertebrate organisms with complementary strengths as models for Development. • Many regulators found in worms or flies are conserved in vertebrates and are associated with human health and disease.. [ABSTRACT FROM AUTHOR]

Lipid droplets (LDs) are lipid-rich organelles universally found in most cells. They serve as a key energy reservoir, actively participate in signal transduction and dynamically communicate with other organelles. LD dysfunction has been associated with a variety of diseases. The content level, composition and mobility of LDs are crucial for their physiological and pathological functions, and these different parameters of LDs are subject to regulation by genetic factors and environmental inputs. Coherent Raman scattering (CRS) microscopy utilizes optical nonlinear processes to probe the intrinsic chemical bond vibration, offering label-free, quantitative imaging of lipids in vivo with high chemical specificity and spatiotemporal resolution. In this Review, we provide an overview over the principle of CRS microscopy and its application in tracking different parameters of LDs in live cells and organisms. We also discuss the use of CRS microscopy in genetic screens to discover lipid regulatory mechanisms and in understanding disease-related lipid pathology. [ABSTRACT FROM AUTHOR]

Carotenoids comprise the most widely distributed natural pigments. In plants, they play indispensable roles in photosynthesis, furnish colors to flowers and fruit and serve as precursor molecules for the synthesis of apocarotenoids, including aroma and scent, phytohormones and other signaling molecules. Dietary carotenoids are vital to human health as a source of provitamin A and antioxidants. Hence, the enormous interest in carotenoids of crop plants. Over the past three decades, the carotenoid biosynthesis pathway has been mainly deciphered due to the characterization of natural and induced mutations that impair this process. Over the year, numerous mutations have been studied in dozens of plant species. Their phenotypes have significantly expanded our understanding of the biochemical and molecular processes underlying carotenoid accumulation in crops. Several of them were employed in the breeding of crops with higher nutritional value. This compendium of all known random and targeted mutants available in the carotenoid metabolic pathway in plants provides a valuable resource for future research on carotenoid biosynthesis in plant species. [ABSTRACT FROM AUTHOR]

Carotenoids comprise the most widely distributed natural pigments. In plants, they play indispensable roles in photosynthesis, furnish colors to flowers and fruit and serve as precursor molecules for the synthesis of apocarotenoids, including aroma and scent, phytohormones and other signaling molecules. Dietary carotenoids are vital to human health as a source of provitamin A and antioxidants. Hence, the enormous interest in carotenoids of crop plants. Over the past three decades, the carotenoid biosynthesis pathway has been mainly deciphered due to the characterization of natural and induced mutations that impair this process. Over the year, numerous mutations have been studied in dozens of plant species. Their phenotypes have significantly expanded our understanding of the biochemical and molecular processes underlying carotenoid accumulation in crops. Several of them were employed in the breeding of crops with higher nutritional value. This compendium of all known random and targeted mutants available in the carotenoid metabolic pathway in plants provides a valuable resource for future research on carotenoid biosynthesis in plant species.

SUMMARYFunctional genomics is the use of systematic gene perturbation approaches to determine the contributions of genes under conditions of interest. Although functional genomic strategies have been used in bacteria for decades, recent studies have taken advantage of CRISPR (clustered regularly interspaced short palindromic repeats) technologies, such as CRISPRi (CRISPR interference), that are capable of precisely modulating expression of all genes in the genome. Here, we discuss and review the use of CRISPRi and related technologies for bacterial functional genomics. We discuss the strengths and weaknesses of CRISPRi as well as design considerations for CRISPRi genetic screens. We also review examples of how CRISPRi screens have defined relevant genetic targets for medical and industrial applications. Finally, we outline a few of the many possible directions that could be pursued using CRISPR-based functional genomics in bacteria. Our view is that the most exciting screens and discoveries are yet to come., Competing Interests: Jason M. Peters has filed for patents related to Mobile-CRISPRi technology and bacterial promoters.

Although nearly 10% of Americans suffer from a rare disease, clinical progress in individual rare diseases is severely compromised by lack of attention and research resources compared to common diseases. It is thus imperative to investigate these diseases at their most basic level to build a foundation and provide the opportunity for understanding their mechanisms and phenotypes, as well as potential treatments. One strategy for effectively and efficiently studying rare diseases is using genetically tractable organisms to model the disease and learn about the essential cellular processes affected. Beyond investigating dysfunctional cellular processes, modeling rare diseases in simple organisms presents the opportunity to screen for pharmacological or genetic factors capable of ameliorating disease phenotypes. Among the small model organisms that excel in rare disease modeling is the nematode Caenorhabditis elegans. With a staggering breadth of research tools, C. elegans provides an ideal system in which to study human disease. Molecular and cellular processes can be easily elucidated, assayed and altered in ways that can be directly translated to humans. When paired with other model organisms and collaborative efforts with clinicians, the power of these C. elegans studies cannot be overstated. This Review highlights studies that have used C. elegans in diverse ways to understand rare diseases and aid in the development of treatments. With continuing and advancing technologies, the capabilities of this small round worm will continue to yield meaningful and clinically relevant information for human health.

Understanding cellular stress response pathways is challenging because of the complexity of regulatory mechanisms and response dynamics, which can vary with both time and the type of stress. We developed a reverse genetic method called ReporterSeq to comprehensively identify genes regulating a stress-induced transcription factor under multiple conditions in a time-resolved manner. ReporterSeq links RNA-encoded barcode levels to pathway-specific output under genetic perturbations, allowing pooled pathway activity measurements via DNA sequencing alone and without cell enrichment or single-cell isolation. We used ReporterSeq to identify regulators of the heat shock response (HSR), a conserved, poorly understood transcriptional program that protects cells from proteotoxicity and is misregulated in disease. Genome-wide HSR regulation in budding yeast was assessed across 15 stress conditions, uncovering novel stress-specific, time-specific, and constitutive regulators. ReporterSeq can assess the genetic regulators of any transcriptional pathway with the scale of pooled genetic screens and the precision of pathway-specific readouts.

Mitochondrial outer membrane ⍺-helical proteins play critical roles in mitochondrial-cytoplasmic communication, but the rules governing the targeting and insertion of these biophysically diverse proteins remain unknown. Here, we first defined the complement of required mammalian biogenesis machinery through genome-wide CRISPRi screens using topologically distinct membrane proteins. Systematic analysis of nine identified factors across 21 diverse ⍺-helical substrates reveals that these components are organized into distinct targeting pathways that act on substrates based on their topology. NAC is required for the efficient targeting of polytopic proteins, whereas signal-anchored proteins require TTC1, a cytosolic chaperone that physically engages substrates. Biochemical and mutational studies reveal that TTC1 employs a conserved TPR domain and a hydrophobic groove in its C-terminal domain to support substrate solubilization and insertion into mitochondria. Thus, the targeting of diverse mitochondrial membrane proteins is achieved through topological triaging in the cytosol using principles with similarities to ER membrane protein biogenesis systems. [Display omitted] • Genome-wide screens identify biogenesis factors for diverse ⍺-helical OMM proteins • Parallel pathways mediate cytosolic targeting based on substrate topology • The NAC complex selectively targets polytopic proteins to the outer membrane • TTC1 is a cytosolic chaperone involved in OMM signal-anchored protein biogenesis Muthukumar et al. use genome-wide and systematic arrayed CRISPRi screens to identify ⍺-helical OMM protein biogenesis and quality control factors that triage substrates by topology. They establish NAC as a targeting factor for polytopic proteins and identify TTC1 as a cytosolic chaperone for signal-anchored proteins, using a C-terminal hydrophobic groove. [ABSTRACT FROM AUTHOR]

Inactivation of the von Hippel Lindau tumor suppressor protein (pVHL) is a hallmark of clear cell Renal Cell Carcinoma (ccRCC), which is the most common form of kidney cancer in adults. In complex with Elongin B/C, pVHL functions as the substrate recognition subunit of a ubiquitin ligase, perhaps best known to target the hypoxia inducible factor (HIF) transcription factor for ubiquitin-dependent proteolysis. Beyond kidney cancer, the pseudo-hypoxic state caused due to chronic HIF activation in pVHL-deficient cells has become a biological model to study hypoxia's profound effects on tumor angiogenesis, metabolism, and epigenetics. However, a number of HIF-independent substrates of pVHL, which function in a broad range of biological pathways, have also been discovered. Independently, the development of high-throughput chemical and genetic screening strategies have enabled the identification of novel, HIF-independent, targetable dependencies in ccRCC. In this review we summarize the history of pVHL and HIF mediated oxygen sensing, discuss the current status of this field, and identify critical challenges that need to be overcome. The confluence of historical discovery, development of unbiased screening strategies, and the evolution of medicinal chemistry has allowed us to begin therapeutically targeting vulnerabilities that emerge due to pVHL loss in ccRCC. Ongoing mechanistic studies on the biological consequences of pVHL loss, therefore, are likely to become the cornerstones of modern therapeutics in renal cancer. [ABSTRACT FROM AUTHOR]

Abstract Background Genome editing by CRISPR-Cas9 technology allows large-scale screening of gene essentiality in cancer. A confounding factor when interpreting CRISPR-Cas9 screens is the high false-positive rate in detecting essential genes within copy number amplified regions of the genome. We have developed the computational tool CRISPRcleanR which is capable of identifying and correcting gene-independent responses to CRISPR-Cas9 targeting. CRISPRcleanR uses an unsupervised approach based on the segmentation of single-guide RNA fold change values across the genome, without making any assumption about the copy number status of the targeted genes. Results Applying our method to existing and newly generated genome-wide essentiality profiles from 15 cancer cell lines, we demonstrate that CRISPRcleanR reduces false positives when calling essential genes, correcting biases within and outside of amplified regions, while maintaining true positive rates. Established cancer dependencies and essentiality signals of amplified cancer driver genes are detectable post-correction. CRISPRcleanR reports sgRNA fold changes and normalised read counts, is therefore compatible with downstream analysis tools, and works with multiple sgRNA libraries. Conclusions CRISPRcleanR is a versatile open-source tool for the analysis of CRISPR-Cas9 knockout screens to identify essential genes.

Identifying microRNA (miRNA) target genes remains a major challenge in understanding the roles miRNAs play in gene regulation. Furthermore, understanding which miRNA–target interactions are the most biologically important is even more difficult. We present CRISPR‐based strategies to identify essential miRNA binding sites. First, CRISPR knockout screens can easily be adapted to identify genes whose inactivation suppresses miRNA mutant phenotypes. Second, a custom approach to target individual miRNA binding sites via CRISPR can identify sites whose mutation recapitulates miRNA mutant phenotypes. We emphasize that the latter approach requires a readout of mutational profile (rather than single guide RNA abundance) when applied in a negative selection setting. Overall, the advent of CRISPR technology alongside improving empirical means of miRNA target identification will accelerate our dissection of miRNA gene regulatory networks. [ABSTRACT FROM AUTHOR]

Senescence is a proliferation arrest that can result from a variety of stresses. Cancer cells can also undergo senescence, but the stresses that provoke cancer cells to undergo senescence are unclear. Here, we use both functional genetic and compound screens in cancer cells harboring a reporter that is activated during senescence to find targets that induce senescence. We show that suppression of the SWI/SNF component SMARCB1 induces senescence in melanoma through strong activation of the MAP kinase pathway. From the compound screen, we identified multiple aurora kinase inhibitors as potent inducers of senescence in RAS mutant lung cancer. Senescent melanoma and lung cancer cells acquire sensitivity to the BCL2 family inhibitor ABT263. We propose a one-two punch approach for the treatment of cancer in which a drug is first used to induce senescence in cancer cells and a second drug is then used to kill senescent cancer cells.

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.

This chapter aims to provide a comprehensive overview of the methodologies available to dissect genetic regulation of the nervous systems in the nematode Caenorhabditis elegans. These techniques encompass genetic screens and genetic tools to unravel the spatial-temporal contribution of genes on neural structure and function. Unbiased genetic screens on random mutations induced by ethyl methanesulfonate (EMS) or target gene silencing by genome-wide RNA interference (RNAi) help progress our understanding of the genetic control of neural development and functions. Complement to unbiased genetic approaches, gene- and protein-targeted manipulation by Cre/LoxP recombination system and auxin-inducible degron (AID) protein degradation system, respectively, helps identify tissues/cells and the time window critical for gene and protein function during the proper execution of a particular behavior. Considering the remarkable conservation of genetic pathways between C. elegans and mammalian systems, elucidating the genetic underpinnings of neural functions and learning behaviors in C. elegans may furnish invaluable insights into analogous processes in more complex organisms. As shown in the following chapter, leveraging these diverse methodologies enable researchers to elucidate the intricate network governing neural function and structure, laying the foundation for innovating strategies to ameliorate cognitive alterations., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Limited translational genomic research data have been reported on the application of exome sequencing and parallel gene testing for preconception carrier screening (PCS). Here, we present individual-level data from a large PCS program in which exome sequencing was routinely performed on either gamete donors (5,845) or infertile patients (8,280) undergoing in vitro fertilization (IVF) treatment without any known family history of inheritable genetic conditions. Individual-level data on pathogenic variants were used to define conditions for PCS based on criteria for severity, penetrance, inheritance pattern, and age of onset. Fetal risk was defined based on actual carrier frequency data accounting for the specific inheritance pattern (fetal disease risk, FDR). In addition, large-scale application of exome sequencing for PCS allowed a deep investigation of the incidence of medically actionable secondary findings in this population. Exome sequencing achieved remarkable clinical sensitivity for reproductive risk of highly penetrant childhood-onset disorders (1/337 conceptions) through analysis of 114 selected gene-condition pairs. A significant contribution to fetal disease risk was observed for rare (carrier rate < 1:100) and X-linked conditions (16.7% and 41.2% of total FDR, respectively). Subgroup analysis of 776 IVF couples identified 37 at increased reproductive risk (4.8%; 95% CI = 3.4–6.5). Further, two additional couples had increased risk for very rare conditions when both members of a parental pair were treated as a unit and the search was extended to the entire exome. About 2.3% of participants showed at least one pathogenic variant for genes included in the updated American College of Medical Genetics and Genomics v2.0 list of secondary findings. Gamete donors and IVF couples showed similar carrier burden for both carrier screening and secondary findings, indicating no causal relationship to fertility. These translational research data will facilitate development of more effective PCS strategies that maximize clinical sensitivity with minimal counterproductive effects. [ABSTRACT FROM AUTHOR]

Drug-induced interstitial lung disease (DIILD) is a serious side effect of chemotherapy in cancer patients with an extremely high mortality rate. In this study, to identify genetic variants with greater risk of DIILD, we carried out whole genome sequencing (WGS) of germline DNA samples from 26 patients who developed DIILD, and conducted a case-control association study between these 26 cases and general Japanese population controls registered in the integrative Japanese Genome Variation Database (iJGVD) as a screening study. The associations of 42 single nucleotide variants (SNVs) showing P < 0.0001 were further validated using an independent cohort of 18 DIILD cases as a replication study. A further combined analysis of the screening and replication studies showed a possible association of two SNVs, rs35198919 in intron 1 of the chromosome 22 open reading frame 34 (C22orf34) and rs12625311 in intron 1 of the teashirt zinc finger homeobox 2 (TSHZ2), with DIILD (Pcombined = 1.87 × 10−5 and 5.16 × 10−5, respectively). Furthermore, in a subgroup analysis of epidermal growth factor receptor (EGFR)–tyrosine kinase inhibitor (TKI)-induced interstitial lung disease (ILD), we observed seven candidate SNVs that were possibly associated with ILD (P < 0.00001). This is the first study to identify genetic markers for the risk of DIILD using WGS. Collectively, our novel findings indicate that these SNVs may be applicable for predicting the risk of DIILD in patients receiving chemotherapy. [ABSTRACT FROM AUTHOR]

Drug resistance is a rapidly emerging concern, thus prompting the development of novel therapeutics or combinatorial therapy. Currently, combinatorial therapy targets are based on knowledge of drug mode of action and/or resistance mechanisms, constraining the number of target proteins. Unbiased genome-wide screens could reveal novel genetic components within interaction networks as potential targets in combination therapies. Testing this, in the context of antimicrobial resistance, we implemented an unbiased genome-wide screen, performed in Saccharomyces cerevisiae expressing a Candida glabrata PDR1+ gain-of-function allele. Gain-of-function mutations in this gene are the principal mediators of fluconazole resistance in this human fungal pathogen. Eighteen synthetically lethal S. cerevisiae genetic mutants were identified in cells expressing C. glabrata PDR1+. One mutant, lacking the histone acetyltransferase Gcn5, was investigated further. Deletion or drug-mediated inhibition of Gcn5 caused a lethal phenotype in C. glabrata cells expressing PDR1+ alleles. Moreover, deletion or drug-mediated inactivation of Gcn5, inhibited the emergence of fluconazole-resistant C. glabrata isolates in evolution experiments. Thus, taken together, the data generated in this study provides proof of concept that synthetically lethal genetic screens can identify novel candidate proteins that when therapeutically targeted could allow effective treatment of drug-resistant infections. [ABSTRACT FROM AUTHOR]

There is a need to investigate and better understand the inherited risk of cancer to ensure that clinical applications provide more accurate assessments and management strategies. Developing research-based next-generation sequencing gene panels that not only target (present-day) clinically actionable susceptibility genes but also genes that currently lack sufficient evidence for risk as well as candidate genes, such as those in DNA repair pathways, can help aid this effort. Therefore, gene panel B.O.P. (reast, varian, and rostate) was developed to evaluate the genetic risk of breast, ovarian and/or prostate cancer, and this manuscript serves as an introduction to B.O.P. and highlights its initial analytical validity assessment. B.O.P targets 87 genes that have been suggested, predicted, or clinically proven to be associated with breast, ovarian, and/or prostate cancer risk using Agilent Technologies Haloplex probes. The probes were designed for 100 base pair reads on an Illumina platform and target both coding and non-coding exons as well as 10 intronic base pairs flanking the intron-exon boundaries. The initial B.O.P screening involved 43 individuals from the Alabama Hereditary Cancer Cohort, and an average sequencing depth of 809X was obtained. Upon variant filtering and validation, true positives had an average sequencing depth of 659X and allele balance of 0.51. The average false positive sequencing depth was 34X and allele balance was 0.33. Although low sequencing depth was not always indicative of a false positive, high sequencing depths (>100X) signified a true positive. Furthermore, sensitivity and specificity of BRCA1/2 were calculated to be 100% and 92.3%, respectively. Overall, this screening enabled the establishment of criteria to alleviate future validation efforts and strongly supports the use of B.O.P. to further elucidate hereditary cancer susceptibility. Ultimately, continued B.O.P. screening will provide insights toward the genetic risk of and overlap between breast, ovarian, and/or prostate cancer. [ABSTRACT FROM AUTHOR]

Mutagenic screening is powerful for identifying key genes involved in developmental processes. However, such screens are successful only in lower organisms. Here, we develop a targeted genetic screening approach in mice through combining androgenetic haploid embryonic stem cells (AG-haESCs) and clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 (CRISPR-Cas9) technology. We produced a mutant semi-cloned (SC) mice pool by oocyte injection of AG-haESCs carrying constitutively expressed Cas9 and an single guide RNA (sgRNA) library targeting 72 preselected genes in one step and screened for bone-development–related genes through skeletal analysis at birth. This yielded 4 genes: Zic1 and Clec11a, which are required for bone development, and Rln1 and Irx5, which had not been previously considered. Whereas Rln1−/− mice exhibited small skeletal size only at birth, Irx5−/− mice showed skeletal abnormalities both in postnatal and adult phases due to decreased bone mass and increased bone marrow adipogenesis. Mechanistically, iroquois homeobox 5 (IRX5) promotes osteoblastogenesis and inhibits adipogenesis by suppressing peroxisome proliferator activated receptor γ (PPARγ) activation. Thus, AG-haESC-mediated functional mutagenic screening opens new avenues for genetic interrogation of developmental processes in mice. [ABSTRACT FROM AUTHOR]

Objective: Non-invasive prenatal testing (NIPT) based on cell-free fetal DNA (cffDNA) is highly accurate in the detection of common fetal autosomal trisomies. Aim of this project was to investigate short-term costs and clinical outcomes of the contingent use of cffDNA for prenatal screening of trisomies 21, 18, 13 within a national health service (NHS). Methods: An economic analysis was developed from the perspective of the Italian NHS to compare two possible scenarios for managing pregnant women: women managed according to the Standard of Care screening (SoC) vs a cffDNA scenario, where Harmony Prenatal Test was introduced as a second line screening choice for women with an “at risk” result from SoC screening. Results: The introduction of cffDNA as a second line screening test, conditional to a risk ≥ 1:1,000 from SoC screening, showed a 3% increase in the detection of trisomies, with a 71% decrease in the number of invasive tests performed. Total short-term costs (pregnancy management until childbirth) decreased by € 19 million (from € 84.5 to 65.5 million). Conclusion: The adoption of the Harmony Prenatal Test in women resulting at risk from SoC screening, implied a greater number of trisomies detection, together with a reduction of the healthcare costs. [ABSTRACT FROM AUTHOR]

Borrelia burgdorferi, the causative agent of Lyme disease in humans, is maintained in a complex biphasic life cycle, which alternates between tick and vertebrate hosts. To successfully survive and complete its enzootic cycle, B. burgdorferi adapts to diverse hosts by regulating genes required for survival in specific environments. Here we describe the first ever use of transposon insertion sequencing (Tn-seq) to identify genes required for B. burgdorferi survival in its tick host. We found that insertions into 46 genes resulted in a complete loss of recovery of mutants from larval Ixodes ticks. Insertions in an additional 56 genes resulted in a >90% decrease in fitness. The screen identified both previously known and new genes important for larval tick survival. Almost half of the genes required for survival in the tick encode proteins of unknown function, while a significant portion (over 20%) encode membrane-associated proteins or lipoproteins. We validated the results of the screen for five Tn mutants by performing individual competition assays using mutant and complemented strains. To better understand the role of one of these genes in tick survival, we conducted mechanistic studies of bb0017, a gene previously shown to be required for resistance against oxidative stress. In this study we show that BB0017 affects the regulation of key borrelial virulence determinants. The application of Tn-seq to in vivo screening of B. burgdorferi in its natural vector is a powerful tool that can be used to address many different aspects of the host pathogen interaction. [ABSTRACT FROM AUTHOR]

Background: Every year more than 800 patients in Denmark are diagnosed with renal cell carcinoma (RCC) of which 3–5% are expected to be part of a hereditary renal cancer syndrome. We performed genetic screening of causative and putative RCC-genes (VHL, FH, FLCN, MET, SDHB, BAP1, MITF, CDKN2B) in RCC-patients suspected of a genetic predisposition. Methods: The cohort consisted of forty-eight Danish families or individuals with early onset RCC, a family history of RCC, a family history of RCC and melanoma or both RCC- and melanoma diagnosis in the same individual. DNA was extracted from peripheral blood samples or cancer-free formalin-fixed paraffin-embedded tissue. Results: One start codon variant of unknown clinical significance (VUS) (c.3G>A, p.Met1Ile) and one missense VUS (c.631A>C, p.Met211Leu) was found in VHL in a patient with RCC-onset at twenty-eight years of age but without other manifestations or family history of von Hippel-Lindau (VHL). Furthermore, in three families we found three different variants in BAP1, one of which was a novel non-segregating missense variant (c.1502G>A, p.Ser501Asn) in a family with two brothers affected with RCC. Finally, we found the known E318K-substitution in MITF in a RCC-affected member of a family with multiple melanomas. No variants were detected in CDKN2B. Conclusion: Although we did find three VUS’s in BAP1 in three families and a pathogenic variant in MITF in one family, pathogenic germline variants in BAP1, MITF or CDKN2B are not frequent causes of hereditary renal cancer in Denmark. It is possible that the high prevalence of risk factors such as male gender, smoking and obesity has influenced the development of cancer in the patients of the current study. Further investigations into putative predisposing genes and risk factors of RCC are necessary to enable better prediction of renal cancer risk or presymptomatic testing of relatives in hereditary renal cancer families. [ABSTRACT FROM AUTHOR]

Drosophila provides an inexpensive and quantitative platform for measuring whole animal drug response. A complementary approach is virtual screening, where chemical libraries can be efficiently screened against protein target(s). Here, we present a unique discovery platform integrating structure-based modeling with Drosophila biology and organic synthesis. We demonstrate this platform by developing chemicals targeting a Drosophila model of Medullary Thyroid Cancer (MTC) characterized by a transformation network activated by oncogenic dRetM955T. Structural models for kinases relevant to MTC were generated for virtual screening to identify unique preliminary hits that suppressed dRetM955T-induced transformation. We then combined features from our hits with those of known inhibitors to create a ‘hybrid’ molecule with improved suppression of dRetM955T transformation. Our platform provides a framework to efficiently explore novel kinase inhibitors outside of explored inhibitor chemical space that are effective in inhibiting cancer networks while minimizing whole body toxicity. [ABSTRACT FROM AUTHOR]

The identification of genes essential for bacterial growth and survival represents a promising strategy for the discovery of antimicrobial targets. Essential genes can be identified on a genome-scale using transposon mutagenesis approaches; however, variability between screens and challenges with interpretation of essentiality data hinder the identification of both condition-independent and condition-dependent essential genes. To illustrate the scope of these challenges, we perform a large-scale comparison of multiple published Pseudomonas aeruginosa gene essentiality datasets, revealing substantial differences between the screens. We then contextualize essentiality using genome-scale metabolic network reconstructions and demonstrate the utility of this approach in providing functional explanations for essentiality and reconciling differences between screens. Genome-scale metabolic network reconstructions also enable a high-throughput, quantitative analysis to assess the impact of media conditions on the identification of condition-independent essential genes. Our computational model-driven analysis provides mechanistic insight into essentiality and contributes novel insights for design of future gene essentiality screens and the identification of core metabolic processes. [ABSTRACT FROM AUTHOR]

Hearing loss (HL) is a common sensory disorder. More than half of HL cases can be attributed to genetic causes. There is no effective therapy for genetic HL at present, early diagnosis to reduce the incidence of genetic HL is important for clinical intervention in genetic HL. Previous studies have identified 111 nonsyndromic hearing loss genes. The most frequently mutated genes identified in NSHL patients in China include GJB2, SLC26A4, and the mitochondrial gene MT-RNR1. It is important to develop HL gene panels in Chinese population, which allow for etiologic diagnosis of both SHL and NSHL. In this study, a total of 220 unrelated Han Chinese patients with bilateral progressive SNHL and 50 unrelated healthy controls were performed Single nucleotide polymorphism (SNP) genotyping using an improved multiplex ligation detection reaction (iMLDR) technique, is to simultaneously detect a total of 32 mutations in ten HL genes, covering all currently characterized mutations involved in the etiology of nonsyndromic or syndromic hearing loss in the Chinese population. The 49 positive samples with known mutations were successfully detected using the iMLDR Technique. For 171 SNHL patients, gene variants were found in 57 cases (33.33%), among which, 30 patients carried mutations in GJB2, 14 patients carried mutations in SLC26A4, seven patients carried mutations in GJB3, and six patients carried mutations in MT-RNR1. The molecular etiology of deafness was confirmed in 12.9% (22/171) of patients carried homozygous variants. These results were verified by Sanger sequencing, indicating that the sensitivity and specificity of the iMLDR technique was 100%. We believe that the implementation of this population-specific technology at an efficient clinical level would have great value in HL diagnosis and treatment. [ABSTRACT FROM AUTHOR]

Adult-onset hearing loss is very common, but we know little about the underlying molecular pathogenesis impeding the development of therapies. We took a genetic approach to identify new molecules involved in hearing loss by screening a large cohort of newly generated mouse mutants using a sensitive electrophysiological test, the auditory brainstem response (ABR). We review here the findings from this screen. Thirty-eight unexpected genes associated with raised thresholds were detected from our unbiased sample of 1,211 genes tested, suggesting extreme genetic heterogeneity. A wide range of auditory pathophysiologies was found, and some mutant lines showed normal development followed by deterioration of responses, revealing new molecular pathways involved in progressive hearing loss. Several of the genes were associated with the range of hearing thresholds in the human population and one, SPNS2, was involved in childhood deafness. The new pathways required for maintenance of hearing discovered by this screen present new therapeutic opportunities. [ABSTRACT FROM AUTHOR]

In order to screen the candidate genes of Fenneropenaeus chinensis related to low-temperature tolerance, this research takes juvenile prawns of F. chinensis (P40) in low temperature stress group (4°C) and normal temperature group (18°C) as experimental materials. The results showed that a total of 127,939 Unigenes with average length of 1,190 bp were obtained by assembly, of which 46% were annotated in the Nr database. A total of 1,698 differentially expressed genes were screened by differential gene expression analysis, of which 920 genes showed up-regulated expression and 778 genes showed down-regulated expression. Both GO and KEGG enrichment analysis revealed that differentially expressed genes were enriched in spliceosomes, ribosomes, bile secretion, ABC transport pathways, and cellular nitrogen compound synthesis. A further in-depth analysis obtained 8 genes that may be associated with low-temperature traits of F. chinensis. Five of them displayed up-regulated expression, including ATP-binding cassette protein C, acid ceramidase, glutathione transferase, C-type lectin and heat shock protein HSP70. The remaining three genes, γ-butyl betaine hydroxylase, β-hexosaminidase A and long chain fatty acid-CoA ligase displayed down-regulated expression. Eight differentially expressed genes were randomly selected and the real time RT-PCR verification showed that their expression levels were consistent with the sequencing results, demonstrating the accuracy of the sequencing results. The results of this study provide basic data for revealing the molecular mechanisms of F. chinensis in response to low temperature stress and the molecular assisted breeding of F. chinensis in low temperature. [ABSTRACT FROM AUTHOR]

Typhoid toxin is a virulence factor for Salmonella Typhi and Paratyphi, the cause of typhoid fever in humans. This toxin has a unique architecture in that its pentameric B subunit, made of PltB, is linked to two enzymatic A subunits, the ADP ribosyl transferase PltA and the deoxyribonuclease CdtB. Typhoid toxin is uniquely adapted to humans, recognizing surface glycoprotein sialoglycans terminated in acetyl neuraminic acid, which are preferentially expressed by human cells. The transport pathway to its cellular targets followed by typhoid toxin after receptor binding is currently unknown. Through a genome-wide CRISPR/Cas9-mediated screen we have characterized the mechanisms by which typhoid toxin is transported within human cells. We found that typhoid toxin hijacks specific elements of the retrograde transport and endoplasmic reticulum-associated degradation machineries to reach its subcellular destination within target cells. Our study reveals unique and common features in the transport mechanisms of bacterial toxins that could serve as the bases for the development of novel anti-toxin therapeutic strategies. [ABSTRACT FROM AUTHOR]

Genetic screens in the nematode Caenorhabditis elegans identified the EGF/Ras and Notch pathways as central for vulval precursor cell fate patterning. Schematically, the anchor cell secretes EGF, inducing the P6.p cell to a primary (1°) vulval fate; P6.p in turn induces its neighbors to a secondary (2°) fate through Delta-Notch signaling and represses Ras signaling. In the nematode Oscheius tipulae, the anchor cell successively induces 2° then 1° vulval fates. Here, we report on the molecular identification of mutations affecting vulval induction in O. tipulae. A single Induction Vulvaless mutation was found, which we identify as a cisregulatory deletion in a tissue-specific enhancer of the O. tipulae lin-3 homolog, confirmed by clustered regularly interspaced short palindromic repeats/Cas9 mutation. In contrast to this predictable Vulvaless mutation, mutations resulting in an excess of 2° fates unexpectedly correspond to the plexin/semaphorin pathway. Hyperinduction of P4.p and P8.p in these mutants likely results from mispositioning of these cells due to a lack of contact inhibition. The third signaling pathway found by forward genetics in O. tipulae is the Wnt pathway; a decrease in Wnt pathway activity results in loss of vulval precursor competence and induction, and 1° fate miscentering on P5.p. Our results suggest that the EGF and Wnt pathways have qualitatively similar activities in vulval induction in C. elegans and O. tipulae, albeit with quantitative differences in the effects of mutation. Thus, the derived induction process in C. elegans with an early induction of the 1° fate appeared during evolution, after the recruitment of the EGF pathway for vulval induction. [ABSTRACT FROM AUTHOR]

Leaf rust (Puccinia triticina Eriks.) is a fungal disease of wheat (Triticum spp.), which causes considerable yield loss. Adult plant resistance (APR) is one of the most sustainable approaches to control leaf rust. In this study, field-testing was carried out across ten different locations, followed by molecular screening, to detect the presence of APR genes, Lr34+, Lr46+, Lr67+ and Lr68 in Indian wheat germplasm. In field screening, 190 wheat accessions were selected from 6,319 accessions based on leaf tip necrosis (LTN), disease severity and the average coefficient of infection. Molecular screening revealed that 73% of the accessions possessed known APR genes either as single or as a combination of two or three genes. The occurrence of increased LTN intensity, decreased leaf rust severity and greater expression of APR genes were more in relatively cooler locations. In 52 lines, although the presence of the APR genes was not detected, it still displayed high levels of resistance. Furthermore, 49 accessions possessing either two or three APR genes were evaluated for stability across locations for grain yield. It emerged that eight accessions had wider adaptability. Resistance based on APR genes, in the background of high yielding cultivars, is expected to provide a high level of race non-specific resistance, which is durable. [ABSTRACT FROM AUTHOR]

Identifying small molecules that inhibit protein synthesis by selectively stalling the ribosome constitutes a new strategy for therapeutic development. Compounds that inhibit the translation of PCSK9, a major regulator of low-density lipoprotein cholesterol, have been identified that reduce LDL cholesterol in preclinical models and that affect the translation of only a few off-target proteins. Although some of these compounds hold potential for future therapeutic development, it is not known how they impact the physiology of cells or ribosome quality control pathways. Here we used a genome-wide CRISPRi screen to identify proteins and pathways that modulate cell growth in the presence of high doses of a selective PCSK9 translational inhibitor, PF-06378503 (PF8503). The two most potent genetic modifiers of cell fitness in the presence of PF8503, the ubiquitin binding protein ASCC2 and helicase ASCC3, bind to the ribosome and protect cells from toxic effects of high concentrations of the compound. Surprisingly, translation quality control proteins Pelota (PELO) and HBS1L sensitize cells to PF8503 treatment. In genetic interaction experiments, ASCC3 acts together with ASCC2, and functions downstream of HBS1L. Taken together, these results identify new connections between ribosome quality control pathways, and provide new insights into the selectivity of compounds that stall human translation that will aid the development of next-generation selective translation stalling compounds to treat disease. [ABSTRACT FROM AUTHOR]

Nuclear size correlates with cell size, but the mechanism by which this scaling is achieved is not known. Here we screen fission yeast gene deletion mutants to identify essential factors involved in this process. Our screen has identified 25 essential factors that alter nuclear size, and our analysis has implicated RNA processing and LINC complexes in nuclear size control. This study has revealed lower and more extreme higher nuclear size phenotypes and has identified global cellular processes and specific structural nuclear components important for nuclear size control. [ABSTRACT FROM AUTHOR]

Endosymbiosis has driven major molecular and cellular innovations. Plasmodium spp. parasites that cause malaria contain an essential, non-photosynthetic plastid—the apicoplast—which originated from a secondary (eukaryote–eukaryote) endosymbiosis. To discover organellar pathways with evolutionary and biomedical significance, we performed a mutagenesis screen for essential genes required for apicoplast biogenesis in Plasmodium falciparum. Apicoplast(−) mutants were isolated using a chemical rescue that permits conditional disruption of the apicoplast and a new fluorescent reporter for organelle loss. Five candidate genes were validated (out of 12 identified), including a triosephosphate isomerase (TIM)-barrel protein that likely derived from a core metabolic enzyme but evolved a new activity. Our results demonstrate, to our knowledge, the first forward genetic screen to assign essential cellular functions to unannotated P. falciparum genes. A putative TIM-barrel enzyme and other newly identified apicoplast biogenesis proteins open opportunities to discover new mechanisms of organelle biogenesis, molecular evolution underlying eukaryotic diversity, and drug targets against multiple parasitic diseases. [ABSTRACT FROM AUTHOR]

The systematic identification of regulatory elements that control gene expression remains a challenge. Genetic screens that use untargeted mutagenesis have the potential to identify protein-coding genes, non-coding RNAs and regulatory elements, but their analysis has mainly focused on identifying the former two. To identify regulatory elements, we conducted a new bioinformatics analysis of insertional mutagenesis screens interrogating WNT signaling in haploid human cells. We searched for specific patterns of retroviral gene trap integrations (used as mutagens in haploid screens) in short genomic intervals overlapping with introns and regions upstream of genes. We uncovered atypical patterns of gene trap insertions that were not predicted to disrupt coding sequences, but caused changes in the expression of two key regulators of WNT signaling, suggesting the presence of cis-regulatory elements. Our methodology extends the scope of haploid genetic screens by enabling the identification of regulatory elements that control gene expression. [ABSTRACT FROM AUTHOR]

Objectives: To explore the molecular epidemiology of rare deafness genes in Taiwanese sensorineural hearing impairment (SNHI) patients with cochlear implantation (CI) by performing massive parallel sequencing (MPS) and correlating genetic factors and CI outcomes. Methods: We enrolled 41 Taiwanese non-syndromic deafness patients with CI that lacked known mutations in common deafness genes. All probands were screened by a targeted exon amplification method that used massively parallel sequencing to screen a customized panel that included 40 relatively rare non-syndromic deafness genes. Results: Thirteen candidate variants in nine relatively rare deafness genes (MYO15A, TMC1, MYH14, MYO3A, ACTG1, COL11A2, DSPP, GRHL2, and WFS1) were identified in 24.4% (10/41) of the non-syndromic deafness probands with CI. According to the ACMG Standards and Guidelines, five variants in MYO15A and ACTG1 were classified as likely pathogenic variants. Two of three multi-generational pedigrees exhibiting deafness were analyzed for the segregation of the disorder with the possible disease-causing variants. Patients with variants detected in most of the identified variant-bearing genes showed relatively good CI outcomes. Conclusions: We successfully identified candidate variants in partially deaf Taiwanese probands who lacked the known mutations in common deafness genes. Comparing the progress of hearing rehabilitation in CI patients with their apparent causative variants and the expression profiles of their altered genes allowed us to speculate on how alterations in specific gene sets may influence outcomes in hearing rehabilitation after CI. [ABSTRACT FROM AUTHOR]

Reverse transcription quantitative real-time PCR (RT-qPCR) is a popular adopted technique to detect gene expression, and the selection of appropriate reference genes is crucial for data normalization. In the present study, seven candidate reference genes were screened to evaluate their expression stability in various flower buds, leaf buds, tissues and cultivars of the English walnut (Juglans regia L.) based on four algorithms (geNorm, Normfinder, Bestkeeper and RefFinder). The results demonstrated that TUA, EF1 and TUB were appropriate reference genes for flower buds at different stages of female flower buds differentiation; TUB and 18S rRNA were best for leaf buds at different stages of female flower buds differentiation; TUB and TUA were suitable for different cultivars; and ACT2, 18S rRNA and GAPDH were useful for different tissues. Moreover, the expression of ACT was not stable among different flower buds, leaf buds and cultivars. The stability of reference genes were confirmed through the analysis of the expression of SPL18 gene. These results will contribute to a reliable normalization of gene expression in J. regia. [ABSTRACT FROM AUTHOR]

Background: Tuberculosis (TB) is the deadliest infectious disease globally. Current case finding approaches may miss many people with TB or detect them too late. Data and methods: This study was a retrospective, spatial analysis of routine TB surveillance and cadastral data in Go Vap district, Ho Chi Minh City. We geocoded TB notifications from 2011 to 2015 and calculated theoretical yields of simulated door-to-door screening in three concentric catchment areas (50m, 100m, 200m) and three notification window scenarios (one, two and four quarters) for each index case. We calculated average yields, compared them to published reference values and fit a GEE (Generalized Estimating Equation) linear regression model onto the data. Results: The sample included 3,046 TB patients. Adjusted theoretical yields in 50m, 100m and 200m catchment areas were 0.32% (95%CI: 0.27,0.37), 0.21% (95%CI: 0.14,0.29) and 0.17% (95%CI: 0.09,0.25), respectively, in the baseline notification window scenario. Theoretical yields in the 50m-catchment area for all notification window scenarios were significantly higher than a reference yield from literature. Yield was positively associated with treatment failure index cases (beta = 0.12, p = 0.001) and short-term inter-province migrants (beta = 0.06, p = 0.022), while greater distance to the DTU (beta = -0.02, p<0.001) was associated with lower yield. Conclusions: This study is an example of inter-departmental collaboration and application of repurposed cadastral data to progress towards the end TB objectives. The results from Go Vap showed that the use of spatial analysis may be able to identify areas where targeted active case finding in Vietnam can help improve TB case detection. [ABSTRACT FROM AUTHOR]