Genome-wide Screening Strategy Research Articles

Genome-Wide Screen for Enhanced Noncanonical Amino Acid Incorporation in Yeast.

Expanding the genetic code beyond the 20 canonical amino acids enables access to a wide range of chemical functionality that is inaccessible within conventionally biosynthesized proteins. The vast majority of efforts to expand the genetic code have focused on the orthogonal translation systems required to achieve the genetically encoded addition of noncanonical amino acids (ncAAs) into proteins. There remain tremendous opportunities for identifying genetic and genomic factors that enhance ncAA incorporation. Here we describe genome-wide screening strategies to identify factors that enable more efficient addition of ncAAs to biosynthesized proteins. These unbiased screens can reveal previously unknown genes or mutations that can enhance ncAA incorporation and deepen our understanding of the translation apparatus.

Whole-genome screens reveal regulators of differentiation state and context-dependent migration in human neutrophils

Neutrophils are the most abundant leukocyte in humans and provide a critical early line of defense as part of our innate immune system. We perform a comprehensive, genome-wide assessment of the molecular factors critical to proliferation, differentiation, and cell migration in a neutrophil-like cell line. Through the development of multiple migration screen strategies, we specifically probe directed (chemotaxis), undirected (chemokinesis), and 3D amoeboid cell migration in these fast-moving cells. We identify a role for mTORC1 signaling in cell differentiation, which influences neutrophil abundance, survival, and migratory behavior. Across our individual migration screens, we identify genes involved in adhesion-dependent and adhesion-independent cell migration, protein trafficking, and regulation of the actomyosin cytoskeleton. This genome-wide screening strategy, therefore, provides an invaluable approach to the study of neutrophils and provides a resource that will inform future studies of cell migration in these and other rapidly migrating cells.

Genetic enhancers of partial PLK1 inhibition reveal hypersensitivity to kinetochore perturbations.

Polo-like kinase 1 (PLK1) is a serine/threonine kinase required for mitosis and cytokinesis. As cancer cells are often hypersensitive to partial PLK1 inactivation, chemical inhibitors of PLK1 have been developed and tested in clinical trials. However, these small molecule inhibitors alone are not completely effective. PLK1 promotes numerous molecular and cellular events in the cell division cycle and it is unclear which of these events most crucially depend on PLK1 activity. We used a CRISPR-based genome-wide screening strategy to identify genes whose inactivation enhances cell proliferation defects upon partial chemical inhibition of PLK1. Genes identified encode proteins that are functionally linked to PLK1 in multiple ways, most notably factors that promote centromere and kinetochore function. Loss of the kinesin KIF18A or the outer kinetochore protein SKA1 in PLK1-compromised cells resulted in mitotic defects, activation of the spindle assembly checkpoint and nuclear reassembly defects. We also show that PLK1-dependent CENP-A loading at centromeres is extremely sensitive to partial PLK1 inhibition. Our results suggest that partial inhibition of PLK1 compromises the integrity and function of the centromere/kinetochore complex, rendering cells hypersensitive to different kinetochore perturbations. We propose that KIF18A is a promising target for combinatorial therapies with PLK1 inhibitors.

RBM33 directs the nuclear export of transcripts containing GC-rich elements.

Although splicing is a major driver of RNA nuclear export, many intronless RNAs are efficiently exported to the cytoplasm through poorly characterized mechanisms. For example, GC-rich sequences promote nuclear export in a splicing-independent manner, but how GC content is recognized and coupled to nuclear export is unknown. Here, we developed a genome-wide screening strategy to investigate the mechanism of export of NORAD, an intronless cytoplasmic long noncoding RNA (lncRNA). This screen revealed an RNA binding protein, RBM33, that directs the nuclear export of NORAD and numerous other transcripts. RBM33 directly binds substrate transcripts and recruits components of the TREX-NXF1/NXT1 RNA export pathway. Interestingly, high GC content emerged as the feature that specifies RBM33-dependent nuclear export. Accordingly, RBM33 directly binds GC-rich elements in target transcripts. These results provide a broadly applicable strategy for the genetic dissection of nuclear export mechanisms and reveal a long-sought nuclear export pathway for transcripts with GC-rich sequences.

Genome-wide activation screens to increase adeno-associated virus production

We describe a genome-wide screening strategy to identify target genes whose modulation increases the capacity of a cell to produce recombinant adeno-associated viral (AAV) vector. Specifically, a single-guide RNA (sgRNA) library for a CRISPR-based genome-wide transcriptional activation screen was inserted into an AAV vector, and iterative rounds of viral infection and rescue in HEK293 producer cells enabled the enrichment of sgRNAs targeting genes whose upregulation increased AAV production. Numerous gain-of-function targets were identified, including spindle and kinetochore associated complex subunit 2 (SKA2) and inositol 1, 4, 5-trisphosphate receptor interacting protein (ITPRIP). Furthermore, individual or combinatorial modulation of these targets in stable producer cell lines increased vector genomic replication and loading into AAV virions, resulting in up to a 3.8-fold increase in AAV manufacturing capacity. Our study offers an efficient approach to engineer viral vector producer cell lines and enhances our understanding of the roles of SKA2 and ITPRIP in AAV packaging.

A pooled genome-wide screening strategy to identify and rank influenza host restriction factors in cell-based vaccine production platforms

Cell-derived influenza vaccines provide better protection and a host of other advantages compared to the egg-derived vaccines that currently dominate the market, but their widespread use is hampered by a lack of high yield, low cost production platforms. Identification and knockout of innate immune and metabolic restriction factors within relevant host cell lines used to grow the virus could offer a means to substantially increase vaccine yield. In this paper, we describe and validate a novel genome-wide pooled CRISPR/Cas9 screening strategy that incorporates a reporter virus and a FACS selection step to identify and rank restriction factors in a given vaccine production cell line. Using the HEK-293SF cell line and A/PuertoRico/8/1934 H1N1 influenza as a model, we identify 64 putative influenza restriction factors to direct the creation of high yield knockout cell lines. In addition, gene ontology and protein complex enrichment analysis of this list of putative restriction factors offers broader insights into the primary host cell determinants of viral yield in cell-based vaccine production systems. Overall, this work will advance efforts to address the public health burden posed by influenza.

Immuno-informatics Analysis to Identify Novel Vaccine Candidates and Design of a Multi-Epitope Based Vaccine Candidate Against Theileria parasites.

Theileriosis poses a serious threat to ruminants in tropical and subtropical countries. It is a tick-borne disease, caused by an apicomplexan parasite, Theileria. The high disease burden in animals causes huge economic losses to marginal farmers. Further, with increasing cases of resistance to commonly used drugs, it is highly desirable to develop better and cost-effective vaccines against theileriosis. The only available vaccine, live attenuated parasite vaccine, has many drawbacks and hence is unsuitable for controlling this disease. Immuno-informatics has emerged as a useful tool in down selection of potential molecules for vaccine development. In this study, we have used an immuno-informatics driven genome-wide screening strategy to identify potential vaccine targets containing important and effective dominant immunogens against Theileria. The proteome of Theileria annulata was screened for proteins with probability of plasma membrane localization or GPI anchor. The proteins non-homologous to the host (bovine) were selected and their antigenicity was analyzed. The B-cell epitopes were identified in the selected proteins and mapped in the modeled structure of the proteins. A total of 19 linear epitopes in 12 proteins, exposed in the extracellular space and having the potential to induce protective antibodies were obtained. Additionally, CTL epitopes which are peptides with 9-mer core sequence, were also identified, modeled and docked with bovine MHC-I structures. The CTL epitopes showing high binding energy with the bovine MHC-I were further engineered in silico to design a putative multi-epitope vaccine candidate against Theileria parasites. The docking studies and molecular dynamics studies with the predicted multi-epitope vaccine candidate and modeled bovine TLR4 exhibited strong binding energy, suggesting that the complex is stable and the putative multi-epitope vaccine candidate can be a potentially good candidate for vaccine development.

Novel Plasmodium antigens identified via genome-based antibody screen induce protection associated with polyfunctional T cell responses

The development of vaccines against complex intracellular pathogens, such as Plasmodium spp., where protection is likely mediated by cellular immune responses, has proven elusive. The availability of whole genome, proteome and transcriptome data has the potential to advance rational vaccine development but yet there are no licensed vaccines against malaria based on antigens identified from genomic data. Here, we show that the Plasmodium yoelii orthologs of four Plasmodium falciparum proteins identified by an antibody-based genome-wide screening strategy induce a high degree of sterile infection-blocking protection against sporozoite challenge in a stringent rodent malaria model. Protection increased in multi-antigen formulations. Importantly, protection was highly correlated with the induction of multifunctional triple-positive T cells expressing high amounts of IFN-γ, IL-2 and TNF. These data demonstrate that antigens identified by serological screening are targets of multifunctional cellular immune responses that correlate with protection. Our results provide experimental validation for the concept of rational vaccine design from genomic sequence data.

Epitope-Based Vaccine Target Screening against Highly Pathogenic MERS-CoV: An In Silico Approach Applied to Emerging Infectious Diseases.

Middle East respiratory syndrome coronavirus (MERS-CoV) with pandemic potential is a major worldwide threat to public health. However, vaccine development for this pathogen lags behind as immunity associated with protection is currently largely unknown. In this study, an immunoinformatics-driven genome-wide screening strategy of vaccine targets was performed to thoroughly screen the vital and effective dominant immunogens against MERS-CoV. Conservancy and population coverage analysis of the epitopes were done by the Immune Epitope Database. The results showed that the nucleocapsid (N) protein of MERS-CoV might be a better protective immunogen with high conservancy and potential eliciting both neutralizing antibodies and T-cell responses compared with spike (S) protein. Further, the B-cell, helper T-cell and cytotoxic T lymphocyte (CTL) epitopes were screened and mapped to the N protein. A total of 15 linear and 10 conformal B-cell epitopes that may induce protective neutralizing antibodies were obtained. Additionally, a total of 71 peptides with 9-mer core sequence were identified as helper T-cell epitopes, and 34 peptides were identified as CTL epitopes. Based on the maximum HLA binding alleles, top 10 helper T-cell epitopes and CTL epitopes that may elicit protective cellular immune responses against MERS-CoV were selected as MERS vaccine candidates. Population coverage analysis showed that the putative helper T-cell epitopes and CTL epitopes could cover the vast majority of the population in 15 geographic regions considered where vaccine would be employed. The B- and T-cell stimulation potentials of the screened epitopes is to be further validated for their efficient use as vaccines against MERS-CoV. Collectively, this study provides novel vaccine target candidates and may prompt further development of vaccines against MERS-CoV and other emerging infectious diseases.

UGPA: unified Gene Pathway Analyzer package for high-throughput genome-wide screening data provides mechanistic overview on human diseases

BackgroundGene set or pathway analysis (GPA) can provide a comprehensive mechanistic overview in delineating molecular pathoetiology of human diseases. Existing tools that can handle for GPA on both mutational and gene expressional aspects are limited. This leads to the development of uGPA (unified Gene Pathway Analyzer). MethodsuGPA package provides a unified solution to knowledge base-driven competitive GPA that can analyze genome-wide screening data derived from multiple platforms (next generation sequencing or microarray) and sample sources (genomic or transcriptomic) on both mutational and gene expressional perspectives. It allows for a quick assessment of perspective outline of gene sets on the studied disease through enrichment test modeled by cumulative hypergeometric distribution and reports the gene components detected with events (mutation or differential gene expression) in the gene sets. ResultsuGPA package has been successfully verified to be applicable on input data generated from NGS-based platforms (transcriptome sequencing and whole-exome sequencing). Genome-wide screening data from other platforms should also apply. ConclusionsuGPA package delivers a simple, flexible and platform-independent functionality to multiple aspects of GPA. It equips clinicians and scientists with a useful tool in studying molecular mechanism of human diseases using modern high-throughput genome-wide screening strategy, which assists personalized therapy.

FSH receptor genotype does not predict metaphase-II oocyte output or fertilization rates in ICSI patients

The objective of this study was to assess the role of the variant p.Asn680Ser in the FSH receptor gene (FSHR) in determining oocyte maturity. It also assessed the relationship between this FSHR variant with metaphase-II oocyte output rate (MOR) and the fertilization rate. This was a prospective observational study based at a tertiary referral centre for reproductive medicine. Women (n=212) undergoing their first cycle of ovarian stimulation for IVF with intracytoplasmic sperm injection (ICSI) were included in the study. Baseline pelvic ultrasound and blood tests were taken on day 2 or 3 of the cycle for assessment of baseline hormones and for DNA extraction. Genotypes for FSHR p.Asn680Ser was determined using TaqMan allelic discrimination assay. The outcome measures were the total dose of exogenous gonadotrophins used, antral follicle count (AFC), number of mature (metaphase-II) oocytes retrieved, MOR and fertilization rate. No statistically significant differences were found between the number of mature oocytes retrieved, MOR or fertilization rates among the patients with different p.Asn680Ser FSHR genotypes. No significant difference was noted in the clinical pregnancy rates per transfer. There is no evidence that the p.Asn680Ser FSHR genotype predicts oocyte maturity.IVF is a complex treatment for infertility requiring costly drugs and carrying significant risk of complications. The procedure aims to stimulate the ovaries to produce more eggs, and conventional methods include a combination of hormones to induce follicle growth, from which eggs are collected. Before beginning the first IVF cycle, knowledge of individual ovarian activity may help predict the response to stimulation and allow clinicians to tailor treatments. Over the last 50years, it has become evident that studying the genetic mechanisms behind variations in drug response in can help control and minimise adverse effects of drugs and their related actions. Hypothetically, a genetic test looking at the variations in the FSH receptor gene may help identify the dosage required and can predict the quality of egg and embryo. One of these variants (at position 680 of exon 10) has been linked to differences in FSH action during ovarian stimulation. None of the studies to date have looked at the role of these genetic markers in the prediction of the quality of eggs and fertilization. We undertook a large prospective study and did not see a relationship between a FSH receptor gene variation and the dosage of gonadotrophins used, quality of eggs or fertilization rates. We surmise that the outcome of ovarian stimulation could be dependent on several factors – both genetic and non-genetic – in a complex multi-factorial model. In the future, genome-wide screening strategies may allow the discovery of further genetic variants associated with ovarian response to stimulation.

Fingering Modulators of Retinoic Acid Signaling Identifies New Prognostic Marker for Neuroblastoma

Properly designed genome-wide screening strategies can provide new insights into biological processes and/or biomarkers for malignant diseases. In this issue of Cancer Cell, Huang et al. demonstrate that the Krüppel zinc-finger protein ZNF423 is critical for retinoic acid signaling and is likely a favorable prognostic marker for neuroblastoma.

Gene discovery through imaging genetics: identification of two novel genes associated with schizophrenia

We have discovered two genes, RSRC1 and ARHGAP18, associated with schizophrenia and in an independent study provided additional support for this association. We have both discovered and verified the association of two genes, RSRC1 and ARHGAP18, with schizophrenia. We combined a genome-wide screening strategy with neuroimaging measures as the quantitative phenotype and identified the single nucleotide polymorphisms (SNPs) related to these genes as consistently associated with the phenotypic variation. To control for the risk of false positives, the empirical P-value for association significance was calculated using permutation testing. The quantitative phenotype was Blood-Oxygen-Level Dependent (BOLD) Contrast activation in the left dorsal lateral prefrontal cortex measured during a working memory task. The differential distribution of SNPs associated with these two genes in cases and controls was then corroborated in a larger, independent sample of patients with schizophrenia (n=82) and healthy controls (n=91), thus suggesting a putative etiological function for both genes in schizophrenia. Up until now these genes have not been linked to any neuropsychiatric illness, although both genes have a function in prenatal brain development. We introduce the use of functional magnetic resonance imaging activation as a quantitative phenotype in conjunction with genome-wide association as a gene discovery tool.

A genome-wide RNAi screen for Wnt/β-catenin pathway components identifies unexpected roles for TCF transcription factors in cancer

The Wnt family of secreted proteins coordinate cell fate decision-making in a broad range of developmental and homeostatic contexts. Corruption of Wnt signal transduction pathways frequently results in degenerative diseases and cancer. We have used an iterative genome-wide screening strategy that employs multiple nonredundant RNAi reagents to identify mammalian genes that participate in Wnt/beta-catenin pathway response. Among the genes that were assigned high confidence scores are two members of the TCF/LEF family of DNA-binding proteins that control the transcriptional output of the pathway. Surprisingly, we found that the presumed cancer-promoting gene TCF7L2 functions instead as a transcriptional repressor that restricts colorectal cancer (CRC) cell growth. Mutations in TCF7L2 identified from cancer genome sequencing efforts abolish its ability to function as a transcriptional regulator and result in increased CRC cell growth. We describe a growth-promoting transcriptional program that is likely activated in CRC tumors with compromised TCF7L2 function. Taken together, the results from our screen and studies focused on members of the TCF/LEF gene family refine our understanding of how aberrant Wnt pathway activation sustains CRC growth.

The Calcium-Sensing Receptor Regulates Plasma Membrane Calcium Adenosine Triphosphatase Isoform 2 Activity in Mammary Epithelial Cells: A Mechanism for Calcium-Regulated Calcium Transport into Milk

The calcium-sensing receptor (CaR) regulates transepithelial calcium transport into milk by mammary epithelial cells. Using a genome-wide screening strategy, we identified the plasma membrane calcium ATPase isoform 2 (PMCA2) as a potential downstream target of the CaR. We show that PMCA2 expression in the mouse mammary gland increases during lactation and that PMCA2 is localized solely to the apical plasma membrane of mammary epithelial cells. In milk from deafwaddler mice, which have mutations in the gene encoding PMCA2, calcium concentrations were reduced, confirming its importance in calcium transport into milk. Furthermore, in cultured primary and EpH4 mouse mammary epithelial cells, CaR stimulation up-regulated calcium-dependent ATPase activity in plasma membrane preparations. By small interfering RNA-mediated gene knockdown of PMCA2, we show that PMCA2 accounts for the preponderance of calcium-ATPase activity. We also show that reduction of CaR expression with small interfering RNA eliminates the ability of extracellular calcium to elicit an increase in calcium-dependent ATPase activity in EpH4 cell membranes. These results demonstrate that activation of the CaR increases PMCA2 activity in mouse mammary epithelial cells, providing a mechanism for the regulation of transepithelial calcium transport by calcium in the lactating mouse mammary gland.

Acute adverse reactions associated with angiotensin-converting enzyme inhibitors: genetic factors and therapeutic implications

Angiotensin-converting enzyme inhibitors (ACEIs) have been used in the treatment of various cardiovascular diseases. Despite the therapeutic benefits of ACEIs, there are several reported side effects, including chronic cough, angioedema and anaphylactoid reactions. These adverse events cannot be explained by the vasodilatory effects of this group of medications. Preliminary studies have shown that patients with a history of developing these side effects have a lower activity of an enzyme called aminopeptidase-P. This enzyme has an important role in degrading bradykinin. This defect in enzymatic activity can be partially explained by genetic variation. Using genome-wide screening strategies, the locus (loci), gene(s) and untimely polymorphisms that explain the low enzymatic activity and side effects can be identified.

Prenatal Diagnosis by Array-CGH

Microscopic karyotype analysis of cultured cells has been regarded as the gold standard for prenatal diagnosis for over 30 years. Since the first application of this technique to prenatal testing in the early 1970′s, this procedure has proved to be highly reliable for identifying chromosome copy number abnormalities (aneuploidy) and large structural rearrangements in foetal cells obtained invasively by either amniocentesis or chorionic villus sampling (CVS). Recognising the need for more rapid testing methods which do not require cell culture, fluorescence in situ hybridisation (FISH) and quantitative fluorescence PCR (QF-PCR) have been introduced to this field in order to answer specific diagnostic questions. However, both FISH and QF-PCR suffer the disadvantage in that they are difficult to scale to a comprehensive, genome-wide screen. Array-comparative genomic hybridisation (array-CGH) in contrast is a comprehensive, genome-wide screening strategy for detecting DNA copy number imbalances which can be rapid, less labour-intensive than karyotype banding analysis and is highly amenable to automation. Array-CGH has the potential to be used for prenatal diagnosis and may address many of the limitations of both conventional microscopic cytogenetic analyses and the more recently employed rapid-screening strategies.