Single-gene knockout experiments can fail to reveal function in the context of redundancy, which is frequently observed among duplicated genes (paralogs) with overlapping functions. We discuss the complexity associated with studying paralogs and outline how recent advances in CRISPR will help address the "phenotype gap" and impact biomedical research.
One of the most powerful ways to develop hypotheses regarding biological functions of conserved genes in a given species, such as in humans, is to first look at what is known about function in another species. Model organism databases (MODs) and other resources are rich with functional information but difficult to mine. Gene2Function (G2F) addresses a broad need by integrating information about conserved genes in a single online resource.
One major challenge encountered with interpreting human genetic variants is the limited understanding of the functional impact of genetic alterations on biological processes. Furthermore, there remains an unmet demand for an efficient survey of the wealth of information on human homologs in model organisms across numerous databases. To efficiently assess the large volume of publically available information, it is important to provide a concise summary of the most relevant information in a rapid user-friendly format. To this end, we created MARRVEL (model organism aggregated resources for rare variant exploration). MARRVEL is a publicly available website that integrates information from six human genetic databases and seven model organism databases. For any given variant or gene, MARRVEL displays information from OMIM, ExAC, ClinVar, Geno2MP, DGV, and DECIPHER. Importantly, it curates model organism-specific databases to concurrently display a concise summary regarding the human gene homologs in budding and fission yeast, worm, fly, fish, mouse, and rat on a single webpage. Experiment-based information on tissue expression, protein subcellular localization, biological process, and molecular function for the human gene and homologs in the seven model organisms are arranged into a concise output. Hence, rather than visiting multiple separate databases for variant and gene analysis, users can obtain important information by searching once through MARRVEL. Altogether, MARRVEL dramatically improves efficiency and accessibility to data collection and facilitates analysis of human genes and variants by cross-disciplinary integration of 18 million records available in public databases to facilitate clinical diagnosis and basic research.
Our understanding of the genetic mechanisms that underlie biological processes has relied extensively on loss-of-function (LOF) analyses. LOF methods target DNA, RNA or protein to reduce or to ablate gene function. By analysing the phenotypes that are caused by these perturbations the wild-type function of genes can be elucidated. Although all LOF methods reduce gene activity, the choice of approach (for example, mutagenesis, CRISPR-based gene editing, RNA interference, morpholinos or pharmacological inhibition) can have a major effect on phenotypic outcomes. Interpretation of the LOF phenotype must take into account the biological process that is targeted by each method. The practicality and efficiency of LOF methods also vary considerably between model systems. We describe parameters for choosing the optimal combination of method and system, and for interpreting phenotypes within the constraints of each method.
The FlyRNAi database of the Drosophila RNAi Screening Center (DRSC) and Transgenic RNAi Project (TRiP) at Harvard Medical School and associated DRSC/TRiP Functional Genomics Resources website (http://fgr.hms.harvard.edu) serve as a reagent production tracking system, screen data repository, and portal to the community. Through this portal, we make available protocols, online tools, and other resources useful to researchers at all stages of high-throughput functional genomics screening, from assay design and reagent identification to data analysis and interpretation. In this update, we describe recent changes and additions to our website, database and suite of online tools. Recent changes reflect a shift in our focus from a single technology (RNAi) and model species (Drosophila) to the application of additional technologies (e.g. CRISPR) and support of integrated, cross-species approaches to uncovering gene function using functional genomics and other approaches.
The rapid rise of CRISPR as a technology for genome engineering and related research applications has created a need for algorithms and associated online tools that facilitate design of on-target and effective guide RNAs (gRNAs). Here, we review the state-of-the-art in CRISPR gRNA design for research applications of the CRISPR-Cas9 system, including knockout, activation and inhibition. Notably, achieving good gRNA design is not solely dependent on innovations in CRISPR technology. Good design and design tools also rely on availability of high-quality genome sequence and gene annotations, as well as on availability of accumulated data regarding off-targets and effectiveness metrics. This article is protected by copyright. All rights reserved.
RNA interference (RNAi) is a widely adopted tool for loss-of-function studies but RNAi results only have biological relevance if the reagents are appropriately mapped to genes. Several groups have designed and generated RNAi reagent libraries for studies in cells or in vivo for Drosophila and other species. At first glance, matching RNAi reagents to genes appears to be a simple problem, as each reagent is typically designed to target a single gene. In practice, however, the reagent-gene relationship is complex. Although the sequences of oligonucleotides used to generate most types of RNAi reagents are static, the reference genome and gene annotations are regularly updated. Thus, at the time a researcher chooses an RNAi reagent or analyzes RNAi data, the most current interpretation of the RNAi reagent-gene relationship, as well as related information regarding specificity (e.g., predicted off-target effects), can be different from the original interpretation. Here, we describe a set of strategies and an accompanying online tool, UP-TORR (for Updated Targets of RNAi Reagents; www.flyrnai.org/up-torr), useful for accurate and up-to-date annotation of cell-based and in vivo RNAi reagents. Importantly, UP-TORR automatically synchronizes with gene annotations daily, retrieving the most current information available, and for Drosophila, also synchronizes with the major reagent collections. Thus, UP-TORR allows users to choose the most appropriate RNAi reagents at the onset of a study, as well as to perform the most appropriate analyses of results of RNAi-based studies.
RNA interference (RNAi) leads to sequence-specific knockdown of gene function. The approach can be used in large-scale screens to interrogate function in various model organisms and an increasing number of other species. Genome-scale RNAi screens are routinely performed in cultured or primary cells or in vivo in organisms such as C. elegans. High-throughput RNAi screening is benefitting from the development of sophisticated new instrumentation and software tools for collecting and analyzing data, including high-content image data. The results of large-scale RNAi screens have already proved useful, leading to new understandings of gene function relevant to topics such as infection, cancer, obesity, and aging. Nevertheless, important caveats apply and should be taken into consideration when developing or interpreting RNAi screens. Some level of false discovery is inherent to high-throughput approaches and specific to RNAi screens, false discovery due to off-target effects (OTEs) of RNAi reagents remains a problem. The need to improve our ability to use RNAi to elucidate gene function at large scale and in additional systems continues to be addressed through improved RNAi library design, development of innovative computational and analysis tools and other approaches.
BACKGROUND: Mapping of orthologous genes among species serves an important role in functional genomics by allowing researchers to develop hypotheses about gene function in one species based on what is known about the functions of orthologs in other species. Several tools for predicting orthologous gene relationships are available. However, these tools can give different results and identification of predicted orthologs is not always straightforward. RESULTS: We report a simple but effective tool, the Drosophila RNAi Screening Center Integrative Ortholog Prediction Tool (DIOPT; http://www.flyrnai.org/diopt), for rapid identification of orthologs. DIOPT integrates existing approaches, facilitating rapid identification of orthologs among human, mouse, zebrafish, C. elegans, Drosophila, and S. cerevisiae. As compared to individual tools, DIOPT shows increased sensitivity with only a modest decrease in specificity. Moreover, the flexibility built into the DIOPT graphical user interface allows researchers with different goals to appropriately 'cast a wide net' or limit results to highest confidence predictions. DIOPT also displays protein and domain alignments, including percent amino acid identity, for predicted ortholog pairs. This helps users identify the most appropriate matches among multiple possible orthologs. To facilitate using model organisms for functional analysis of human disease-associated genes, we used DIOPT to predict high-confidence orthologs of disease genes in Online Mendelian Inheritance in Man (OMIM) and genes in genome-wide association study (GWAS) data sets. The results are accessible through the DIOPT diseases and traits query tool (DIOPT-DIST; http://www.flyrnai.org/diopt-dist). CONCLUSIONS: DIOPT and DIOPT-DIST are useful resources for researchers working with model organisms, especially those who are interested in exploiting model organisms such as Drosophila to study the functions of human disease genes.
BACKGROUND: A genomic catalogue of protein-protein interactions is a rich source of information, particularly for exploring the relationships between proteins. Numerous systems-wide and small-scale experiments have been conducted to identify interactions; however, our knowledge of all interactions for any one species is incomplete, and alternative means to expand these network maps is needed. We therefore took a comparative biology approach to predict protein-protein interactions across five species (human, mouse, fly, worm, and yeast) and developed InterologFinder for research biologists to easily navigate this data. We also developed a confidence score for interactions based on available experimental evidence and conservation across species. RESULTS: The connectivity of the resultant networks was determined to have scale-free distribution, small-world properties, and increased local modularity, indicating that the added interactions do not disrupt our current understanding of protein network structures. We show examples of how these improved interactomes can be used to analyze a genome-scale dataset (RNAi screen) and to assign new function to proteins. Predicted interactions within this dataset were tested by co-immunoprecipitation, resulting in a high rate of validation, suggesting the high quality of networks produced. CONCLUSIONS: Protein-protein interactions were predicted in five species, based on orthology. An InteroScore, a score accounting for homology, number of orthologues with evidence of interactions, and number of unique observations of interactions, is given to each known and predicted interaction. Our website http://www.interologfinder.org provides research biologists intuitive access to this data.
RNA interference (RNAi) provides a powerful reverse genetics approach to analyze gene functions both in tissue culture and in vivo. Because of its widespread applicability and effectiveness it has become an essential part of the tool box kits of model organisms such as Caenorhabditis elegans, Drosophila, and the mouse. In addition, the use of RNAi in animals in which genetic tools are either poorly developed or nonexistent enables a myriad of fundamental questions to be asked. Here, we review the methods and applications of in vivo RNAi to characterize gene functions in model organisms and discuss their impact to the study of developmental as well as evolutionary questions. Further, we discuss the applications of RNAi technologies to crop improvement, pest control and RNAi therapeutics, thus providing an appreciation of the potential for phenomenal applications of RNAi to agriculture and medicine.