Stephanie E. Mohr

Genomic Screening with RNAi: Results and Challenges

RNA interference (RNAi) is an effective tool for genome-scale, high-throughput analysis of gene function. In the past five years, a number of genome-scale RNAi high-throughput screens (HTSs) have been done in both Drosophila and mammalian cultured cells to study diverse biological processes, including signal transduction, cancer biology, and host cell responses to infection. Results from these screens have led to the identification of new components of these processes and, importantly, have also provided insights into the complexity of biological systems, forcing new and innovative approaches to understanding functional networks in cells. Here, we review the main findings that have emerged from RNAi HTS and discuss technical issues that remain to be improved, in particular the verification of RNAi results and validation of their biological relevance. Furthermore, we discuss the importance of multiplexed and integrated experimental data analysis pipelines to RNAi HTS.

RNAi screening comes of age: improved techniques and complementary approaches

Gene silencing through sequence-specific targeting of mRNAs by RNAi has enabled genome-wide functional screens in cultured cells and in vivo in model organisms. These screens have resulted in the identification of new cellular pathways and potential drug targets. Considerable progress has been made to improve the quality of RNAi screen data through the development of new experimental and bioinformatics approaches. The recent availability of genome-editing strategies, such as the CRISPR (clustered regularly interspaced short palindromic repeats)–Cas9 system, when combined with RNAi, could lead to further improvements in screen data quality and follow-up experiments, thus promoting our understanding of gene function and gene regulatory networks.

Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9

Significance Using the recently introduced Cas9/sgRNA technique, we have developed a method for specifically targeting Drosophila germ-line cells to generate heritable mutant alleles. We have established transgenic lines that stably express Cas9 in the germ line and compared different promoters and scaffolds of sgRNA in terms of their efficiency of mutagenesis. An overall mutagenesis rate of 74.2% was achieved with this optimized system, as determined by the number of mutant progeny out of all progeny screened. We also evaluated the off-targets associated with the method and established a Web-based resource, as well as a searchable, genome-wide database of predicted sgRNAs appropriate for genome engineering in flies. Our results demonstrate that this optimized Cas9/sgRNA system in Drosophila is efficient, specific, and cost-effective and can be readily applied in a semi-high-throughput manner. The ability to engineer genomes in a specific, systematic, and cost-effective way is critical for functional genomic studies. Recent advances using the CRISPR-associated single-guide RNA system (Cas9/sgRNA) illustrate the potential of this simple system for genome engineering in a number of organisms. Here we report an effective and inexpensive method for genome DNA editing in Drosophila melanogaster whereby plasmid DNAs encoding short sgRNAs under the control of the U6b promoter are injected into transgenic flies in which Cas9 is specifically expressed in the germ line via the nanos promoter. We evaluate the off-targets associated with the method and establish a Web-based resource, along with a searchable, genome-wide database of predicted sgRNAs appropriate for genome engineering in flies. Finally, we discuss the advantages of our method in comparison with other recently published approaches.

RNAi screening: new approaches, understandings, and organisms

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. WIREs RNA 2012, 3:145–158. doi: 10.1002/wrna.110

The Transgenic RNAi Project at Harvard Medical School: Resources and Validation

To facilitate large-scale functional studies in Drosophila, the Drosophila Transgenic RNAi Project (TRiP) at Harvard Medical School (HMS) was established along with several goals: developing efficient vectors for RNAi that work in all tissues, generating a genome-scale collection of RNAi stocks with input from the community, distributing the lines as they are generated through existing stock centers, validating as many lines as possible using RT–qPCR and phenotypic analyses, and developing tools and web resources for identifying RNAi lines and retrieving existing information on their quality. With these goals in mind, here we describe in detail the various tools we developed and the status of the collection, which is currently composed of 11,491 lines and covering 71% of Drosophila genes. Data on the characterization of the lines either by RT–qPCR or phenotype is available on a dedicated website, the RNAi Stock Validation and Phenotypes Project (RSVP, http://www.flyrnai.org/RSVP.html), and stocks are available from three stock centers, the Bloomington Drosophila Stock Center (United States), National Institute of Genetics (Japan), and TsingHua Fly Center (China).

Protein complex-based analysis framework for high-throughput data sets.

An analysis tool maps network dynamics at the protein complex level in multiple species. Complexes Reveal Signaling Dynamics Analysis of high-throughput data sets can provide information about changes in gene expression, protein abundance, and signaling pathway activity. However, current data mining approaches do not identify changes to functional protein complexes within a pathway over time, a critical aspect for network analysis. Vinayagam et al. developed an interactive Web tool called COMPLEAT, which uses raw genome-wide, RNA interference data to map protein complex dynamics during the cellular response to stimuli in humans, flies, and yeast. Using phosphorylated extracellular signal–regulated kinase as a marker for pathway activation, COMPLEAT identified the Brahma complex in the cellular response to insulin, a prediction that was validated in a Drosophila cell line. Analysis of high-throughput data increasingly relies on pathway annotation and functional information derived from Gene Ontology. This approach has limitations, in particular for the analysis of network dynamics over time or under different experimental conditions, in which modules within a network rather than complete pathways might respond and change. We report an analysis framework based on protein complexes, which are at the core of network reorganization. We generated a protein complex resource for human, Drosophila, and yeast from the literature and databases of protein-protein interaction networks, with each species having thousands of complexes. We developed COMPLEAT (http://www.flyrnai.org/compleat), a tool for data mining and visualization for complex-based analysis of high-throughput data sets, as well as analysis and integration of heterogeneous proteomics and gene expression data sets. With COMPLEAT, we identified dynamically regulated protein complexes among genome-wide RNA interference data sets that used the abundance of phosphorylated extracellular signal–regulated kinase in cells stimulated with either insulin or epidermal growth factor as the output. The analysis predicted that the Brahma complex participated in the insulin response.

Identification of potential drug targets for tuberous sclerosis complex by synthetic screens combining CRISPR-based knockouts with RNAi

Combining targeted CRISPR-mediated gene editing with an RNAi-mediated screen identifies candidate drug targets. Double screening for drug targets The tumor suppressors TSC1 and TSC2 form a complex that limits the activity of mTORC1, a multiprotein complex that couples nutrient availability to cell proliferation and growth. Individuals with mutations in either of the TSC-encoding genes develop tumors in various organs. Housden et al. used CRISPR to generate Drosophila cell lines that lacked either TSC1 or TSC2 and performed RNAi screens directed against kinase- and phosphatase-encoding genes in these cell lines to identify those genes that limited the growth of cells lacking either TSC1 or TSC2. Candidate genes that, when knocked down, suppressed the growth of both of the TSC1- and TSC2-deficient Drosophila cell lines (but not that of normal cells) were validated in TSC2-deficient human cells. Because these candidates are evolutionarily conserved, they are more likely to be bona fide drug targets, and this combination of techniques and multispecies screening could be used to identify drug targets for other diseases. The tuberous sclerosis complex (TSC) family of tumor suppressors, TSC1 and TSC2, function together in an evolutionarily conserved protein complex that is a point of convergence for major cell signaling pathways that regulate mTOR complex 1 (mTORC1). Mutation or aberrant inhibition of the TSC complex is common in various human tumor syndromes and cancers. The discovery of novel therapeutic strategies to selectively target cells with functional loss of this complex is therefore of clinical relevance to patients with nonmalignant TSC and those with sporadic cancers. We developed a CRISPR-based method to generate homogeneous mutant Drosophila cell lines. By combining TSC1 or TSC2 mutant cell lines with RNAi screens against all kinases and phosphatases, we identified synthetic interactions with TSC1 and TSC2. Individual knockdown of three candidate genes (mRNA-cap, Pitslre, and CycT; orthologs of RNGTT, CDK11, and CCNT1 in humans) reduced the population growth rate of Drosophila cells lacking either TSC1 or TSC2 but not that of wild-type cells. Moreover, individual knockdown of these three genes had similar growth-inhibiting effects in mammalian TSC2-deficient cell lines, including human tumor-derived cells, illustrating the power of this cross-species screening strategy to identify potential drug targets.

Stringent Analysis of Gene Function and Protein-Protein Interactions Using Fluorescently Tagged Genes

In Drosophila collections of green fluorescent protein (GFP) trap lines have been used to probe the endogenous expression patterns of trapped genes or the subcellular localization of their protein products. Here, we describe a method, based on nonoverlapping, highly specific, shRNA transgenes directed against GFP, that extends the utility of these collections to loss-of-function studies. Furthermore, we used a MiMIC transposon to generate GFP traps in Drosophila cell lines with distinct subcellular localization patterns, which will permit high-throughput screens using fluorescently tagged proteins. Finally, we show that fluorescent traps, paired with recombinant nanobodies and mass spectrometry, allow the study of endogenous protein complexes in Drosophila.

Resources for Functional Genomics Studies in Drosophila melanogaster

Drosophila melanogaster has become a system of choice for functional genomic studies. Many resources, including online databases and software tools, are now available to support design or identification of relevant fly stocks and reagents or analysis and mining of existing functional genomic, transcriptomic, proteomic, etc. datasets. These include large community collections of fly stocks and plasmid clones, “meta” information sites like FlyBase and FlyMine, and an increasing number of more specialized reagents, databases, and online tools. Here, we introduce key resources useful to plan large-scale functional genomics studies in Drosophila and to analyze, integrate, and mine the results of those studies in ways that facilitate identification of highest-confidence results and generation of new hypotheses. We also discuss ways in which existing resources can be used and might be improved and suggest a few areas of future development that would further support large- and small-scale studies in Drosophila and facilitate use of Drosophila information by the research community more generally.

ZIMP ENCODES A HOMOLOGUE OF MOUSE MIZ1 AND PIAS3 AND IS AN ESSENTIAL GENE IN DROSOPHILA MELANOGASTER

The related mouse proteins Miz1 and PIAS3, which have predicted zinc finger domains, interact with the transcription factors Msx2 and STAT3, modulating the ability of Msx2 and STAT3 to regulate transcription. Here, we describe a Drosophila gene, zimp, that encodes a protein with similarity to Miz1 and PIAS3. The zimp gene appears to be post-transcriptionally regulated, as three alternatively spliced forms are detected in a cDNA library screen and on an RNA blot. In addition, all three zimp transcripts are detected in embryonic mRNA, but only two of the transcripts are detected in adult mRNA. The three transcripts have the ability to encode two proteins, of 554 and 522 amino acids. The two Zimp amino acid sequences share an amino-terminal 515-amino-acid region and differ in their carboxy-termini. These proteins and related proteins in other organisms, including mammals, C. elegans, yeast, and plants, share a highly conserved region predicted to form a zinc finger. Deletion of the zimp gene or P-element insertion in zimp is lethal; thus, zimp is an essential gene in Drosophila. These data underscore the potential importance of Zimp-related proteins cross-species, and conservation of the putative zinc finger domain suggests that it is functionally important.