Core Spotlight: Collaborative Functional Genomics
In this DF/HCC eNews Spotlight Edition, we present the DF/HCC Collaborative Functional Genomics Core, which provides a single portal through which DF/HCC members can learn about RNAi and CRISPR-Cas9 screen services relevant to mouse, human, and Drosophila. Understanding the mechanisms that underlie the development and progression of cancer is fundamental to finding better approaches to prevention and treatment. High-throughput RNA interference (RNAi) and CRISPR-Cas9 screening provide state-of-the-art methods for large-scale investigation of gene function, including uncovering of synthetic lethal gene interactions.
CRISPR technologies have emerged as an exciting complement to our continued support for RNAi projects. Our member facilities stay at the leading edge, helping core users apply CRISPR technologies to functional genomics projects. To accomplish this, we both develop approaches in-house in collaboration with core users and connect with relevant reagent companies. Together with the DNA Resource Core and new Mouse Transgenic Core, the Collaborative Functional Genomics Core recently became a member of the Millipore Sigma CRISPR Core Partnership program. Participation in the program gives users of our core facilities access to expert consultation and discounted reagent pricing. Please contact Stephanie Mohr, core director, or individual participating facilities to learn what combination of available technologies will best fit your experimental goals.
With the installation of the IntelliCyt iQUE Screener PLUS at ICCB-Longwood, it is now possible for DF/HCC members to conduct flow cytometry experiments in an automated 96- or 384-well high throughput format.
- Consultation on RNAi and CRISPR screens, and CRISPR-Cas9 engineering projects
- Reagents for functional genomics screening (siRNA, shRNA and other reagent libraries)
- Pooled RNAi screening libraries—human or mouse cells
- Pooled CRISPR-Cas9 screening libraries—human or Drosophila cells
- Arrayed RNAi screening libraries—human cells
- Arrayed RNAi screening libraries—mouse cells
- Arrayed RNAi screening libraries—Drosophila cells
- Stage- and tissue-specific in vivo RNAi screening in Drosophila
- Reagents for miRNA interrogation (mimics and inhibitors)—human cells and Drosophila
- Reagents for lncRNA interrogation (siRNA libraries) – human cells
- Bioinformatics support for RNAi and CRISPR screens, and CRISPR-Cas9 projects, including
- Development of custom gene lists for focused screens
- Statistical and enrichment analysis of large-scale screen data
- Off-target analysis (including via our Online GESS software tool)
- CRISPR-Cas9 knock-out and knock-in project design
- Automated assay read-out equipment (96-well and 384-well compatible)
- Luminescence and fluorescence “plate readers”
- Laser-scanning and flow cytometers
- Epifluorescence microscopes
- Fluorescence confocal, epifluorescence, and bright field microscope (4x, 10x, 20x, 60x)
- Automated liquid handling robotics for screening and other high throughput or automated studies
- Reagents for secondary screens and follow-up studies
- Additional reagents and resources
Cross-species, sensitized cell background approach to identification of potential therapeutic targets for tuberous sclerosis
The collaborating groups recently used CRISPR-Cas technology to knock out the Drosophila orthologs of TSC1 and TSC2 genes, then used a novel RNAi technology called Variable Dose Analysis (VDA) to screen for genes that when knocked down, compromise viability of TSC1 and TSC2 mutant cells but not wild-type cells. This extends previous work by these collaborators to use a cross-species approach to identify conserved proteins known to be targeted by FDA-approved drugs.
Contribution of the Core: The core provided a large-scale library of shRNAs targeting Drosophila orthologs of human genes that encode proteins known to be targeted by FDA-approved drugs. This leveraged an existing core reagent collection and put automation at the core to use in a new way, supporting application of the new VDA screening approach at large scale.
Publication: Housden BE, Li Z, Kelley C, Wang Y, Hu Y, Valvezan AJ, Manning BD, Perrimon N. Improved detection of synthetic lethal interactions in Drosophila cells using variable dose analysis (VDA). Proc Natl Acad Sci U S A. 2017 Nov 28. PMID: 29183982.
A focused siRNA screen to identify cellular regulators of IRF5 signaling
PI: Martin DorfHMS (Cancer Cell Biology, Cancer Immunology Programs)
The Dorf Laboratory conducted a focused siRNA screen of ~3,000 cellular targets to identify proteins that govern TLR7-dependent IRF5 signaling. The results of this experiment, combined with a large-scale proteomics study, enabled the team to identify novel regulators of this innate immune signaling cascade. NXF1 was identified in both screening approaches and demonstrated to form a complex with IRF5 and impact its TLR7-dependent activation.
Contribution of the Core: The Core provided advice and guidance at all stages of the siRNA screening process, including analysis and follow up approaches, as well as the siRNA library screened and laboratory automation.
Publication: Fu B, Zhao M, Wang L, Patil G, Smith JA, Juncadella IJ, Zuvela-Jelaska L, Dorf ME, Li S. RNAi Screen and Proteomics Reveal NXF1 as a Novel Regulator of IRF5 Signaling. Sci Rep 2017 Jun 2. PMID:28578407
A focused siRNA screen to identify super enhancer-associated proteins in neuroblastoma
PI: Jay Bradner (Novartis, formerly of DFCI, and DF/HCC Developmental Therapeutics, Lymphoma and Myeloma Programs)
The lab of Dr. Badner and colleagues identified preferential loading of Brd4 at specific enhancer sites within the genome (Lovén J, Hoke HA, Lin CY, Lau A, Orlando DA, Vakoc CR, Bradner JEDFCI, Lee TI, Young RA. Selective inhibition of tumor oncogenes by disruption of super-enhancers. Cell. 2013 Apr 11;153(2):320-34. PMC3760967). Based upon ChIP-seq data, the Bradner Lab worked with the Core to generate a custom human siRNA library to target proteins that bind to those enhancer sites in neuroblastoma cells, with the goal of identifying potential cancer vulnerabilities for future therapeutic approaches. The siRNAs were screened successfully at ICCB-L and genes required for the viability and proliferation of neuroblastoma cells were preliminarily identified. These results formed the part of the foundation for this recent publication.
Contribution of the Core: The Core generated a custom human siRNA library, provided advice and guidance on assay optimization and screening, and provided the laboratory automation for screening the siRNAs in 384-well format.
Publication: Zeid R, Lawlor MA, Poon E, Reyes JM, Fulciniti M, Lopez MA, Scott TG, Nabet B, Erb MA, Winter GE, Jacobson Z, Polaski DR, Karlin KL, Hirsch RA, Munshi NP, Westbrook TF, Chesler L, Lin CY, Bradner JE. Enhancer invasion shapes MYCN-dependent transcriptional amplification in neuroblastoma. Nat Genet 2018. Jan 29. PMID:29379199
For more information or to contact the Collaborative Functional Genomics Core: