The Victorian Centre for Functional Genomics (VCFG) at Peter Mac enables biomedical researchers Australia-wide the ability to perform novel discovery-based high throughput screens using multiple platforms.
The VCFG facilitates functional interrogation of all genes in the genome, or selected boutique collections using CRISPR/cas9, small interfering RNA (siRNA), micro RNA (miRNA), long non-coding RNA (lncRNA) and short hairpin RNA (shRNA) approaches. The VCFG enables large and small scale drug screens using commercially available compounds or BYO compounds. The VCFG has extensive experience in quantitative high content phenotypic screening and analysis that forms the readout for the majority of the screens.
The VCFG has established an Australia-first, Reverse Phase Protein Array platform, another high throughput discovery technology that allows for rapid quantitation of the expression of native and phospho-specific protein isoforms in very small sample populations.
The high throughput capacity of the VCFG requires automated liquid handling instrumentation (mainframe robot and independent workstations), plate readers and high content microscopy capabilities. The VCFG primarily operates a ‘researcher driven, staff assisted’ model whereby the researcher is embedded in the facility, trained on appropriate equipment and fully supported by the VCFG team from assay concept design, through screening and analysis. Fee for service contract-based screening can also be considered. The VCFG is headed by A/Prof Kaylene Simpson.
A recent revolutionary discovery of the CRISPR/Cas9 system enables gene deletion directly at the genomic level and therefore offers a parallel platform for gene perturbation complementary to RNAi. This approach uses the bacterially derived enzyme Cas9 and a gene-specific single guide RNA molecule (sgRNA) to introduce nicks in precisely defined genomic locations leading to a complete gene knockout.
Pooled viral CRISPR screening
Similarly to shRNA screens, CRISPR can be used in a range of biological contexts, investigating gene function in either positive selection or negative selection screens. Compared with shRNA screens, however, pooled CRISPR screens typically display low levels of off-targets and biological variation making the hit identification easier. The VCFG houses the latest generation of genome-wide pooled CRISPR libraries for human (Brunello library) and mouse (Brie library) screening of protein coding genes. Both libraries contain 4 sgRNA per gene (in total 76,441 sgRNA in the Brunello library and 78,637 sgRNA in the Brie library), and they implement the latest algorithm for optimal sgRNA design enabling highly efficient genome editing. The VCFG also houses both human and mouse pooled activator and inhibitor libraries, CRISPRa (Calabrese and Caprano) and CRISPRi (Docetto and Dolomiti). To identify genes responsible for the phenotype, we use next-generation sequencing, and together with the Molecular Genomics core we offer a full sequencing pipeline and subsequent data analysis and hit identification is fully supported by the VCFG bioinformatics team.
Arrayed synthetic CRISPR screening
The synthetic CRISPR platform offers CRISPR-based gene knockout in an arrayed format coupled with transient transfection performed using similar methodology to siRNA screening in 96 or 384 well format. As with siRNAs, this approach dramatically expands the types of downstream biological assays used to assess the resulting phenotype, and it allows immediate identification of genes responsible for the phenotype, thus simplifying downstream validation steps. This platform uses synthetic purified sgRNAs (comprising crRNA and trRNA), which are transiently delivered into the Cas9-stable cells using lipid transfection reagents, and the phenotype is assessed typically within 72h post-transfection. Compared to the vector-based sgRNA system, the synthetic CRISPR sgRNAs induce a much higher level of editing efficiency, and the phenotypes can be detected without further selection following sgRNA transfection.
The siRNA platform is based on Dharmacon's siGENOME SMARTpool reagents; four sequences to each gene in a single well, arrayed in either 96 or 384 well plate format. siRNAs are transiently introduced into cells using lipid-based reagents and assayed between three to seven days post-transfection and data is analysed using an in-house bioinformatics pipeline to statistically rank the gene list.
Screeners can perform imaging based high throughput, high content cellular analysis, or fluorescence/luminescence based phenotypic quantitation using a high throughput plate reader. Often, researchers multiplex their screens and use both platforms. Recent screens include identification of targets involved in synthetic lethality, drug resistance, cell motility, pathogenic viral entry and regulation of the epithelial to mesenchymal transition of cancer cells.
Using high throughput liquid handling automation the VCFG enables all types of compound screening approaches from cell-based screens to protein complex interaction screens. From straightforward dose curves with BYO drugs and multiple cell lines to large compound collections there is great creativity in this screening approach and we work with you to develop the assay readouts. Compounds are primarily sourced from Compounds Australia
The miRNA platform is based on Dharmacon’s miRIDIAN mimic and inhibitor collections which over-express or knockdown all the miRs in the human genome. The screening strategies are very similar to working with siRNAs, which makes this platform an attractive option to screen following a protein coding SMARTpool screen to enable you to screen the ‘whole-genome’, however, with the clear importance of miRNA regulation of many protein coding genes, many researchers are interested in screening the miRNA collections alone.
In situations where gene deletion is considered less desirable, the VCFG has a pooled shRNA screening platform using a lentiviral shRNA-mir30 vector system (pGIPZ) to enable stable knockdown in primary, non-dividing and standard cell lines. shRNAs are screened in pools with at least five constructs targeting each human gene. The VCFG offers genome-wide and custom screening options and together with the Molecular Genomics core has developed next-generation sequencing pipeline to identify the genes responsible for the biological phenotype of an assay.
Based on the highly sensitive Zeptosens quantitative proteomics platform, researchers can extract protein lysates from cell lines or tissue samples using a proprietary buffer system, submit them to the VCFG and select from over 160 native and phospho- antibodies covering numerous signalling pathways (the collection is continually expanding). Proteins are spotted onto proprietary glass slides called chips in a grid pattern, with 70 protein lysates spotted per grid. Each chip can accommodate 6 grids, which translates to 6 antibodies per chip. Due to the sensitivity of the system, we highly recommend generating lysates from multiple biological replicates (suggest 3 replicates). We suggest all researchers begin with a pilot screen and offer multiplexing capacity to share chips where sample size is small. Large (many samples and screening large numbers of antibodies) or small (small number of samples, limited number of antibodies) scale screens can be performed, the beauty of the system is the inherent flexibility in screening options. All antibodies are verified in at least 3 cell lines before use and we also offer a BYO antibody validation service. We can also run validation westerns subsequent to the RPPA analyses. As with all platforms, the VCFG has a bioinformatics pipeline for quantitative analysis of expression levels and data will be presented in multiple formats in discussion with the researcher.
For more details contact A/Prof Kaylene Simpson. The RPPA platform is co-funded by the University of Melbourne, Melbourne Collaborative Research Infrastructure Program together with partners Bio21, University of Melbourne Department of Anatomy and Neuroscience, Murdoch Childrens Research Institute and Peter Mac.
A recent publication that has utilised the RPPA platform is Pearson et al., Cancer Discovery, June 8(6):764-779, 2018
- human whole-genome CRISPR pooled library (Brunello)
- mouse whole-genome CRISPR pooled library (Brie)
- human whole-genome CRISPRa pooled library (Calabrese)
- mouse whole-genome CRISPRa pooled library (Caprano)
- human whole-genome CRISPRi pooled library (Dolcetto)
- mouse whole-genome CRISPRi pooled library (Dolomiti)
- individual sgRNA constructs for assay development
- Cas9 and dCas9 reporter virus
- 28 independent barcoded primers for library amplification and sample delineation
- human whole genome version 3 shRNAmiR individual shRNAs and viral pools (pGIPZ backbone)
- custom shRNAmiR collections (apoptosis, cell cycle, polarity, kinome, transcription factors, GPCRs, phosphatases)
- mouse whole genome shRNA viral pools (pLKO backbone)
- standard operating procedures for all aspects of the pooled screening process
- next generation single molecule DNA sequencing amplification service
- bioinformatics analysis pipeline
- custom synthetic crRNA libraries
- human synthetic CRISPR kinome
- human and mouse siGENOME SMARTpool and individual duplex siRNA libraries
- human ON-TARGET plus SMARTpool library
- human miRIDIAN miRNA mimic and inhibitor collections
- human Lincode long non-coding RNA library
- comprehensive user guide and instrument documentation.
- aliquots of controls and lipid reagents for assay development
- automated wound healing assay
- Zeptosens lysis buffer
- Over 160 validated antibodies (primarily directed to human target genes raised in rabbit but majority have mouse reactivity)
- Capacity for western blotting validation using the LiCOR Odyssey and accessing the antibody collection
- InCELL western screening capabilities
- high-content microscopy using fully automated Cellomics confocal CX7, Arrayscan VTi and Cell Insight (x2)
- Caliper ALH3000 liquid handling robot for all transfection and speciality liquid handling applications
- JanusG3 liquid handling robot
- BioTek 406: three units that aspirate and dispense media, cells, fix and stain and add drugs
- Cytation5, a sophisticated plate imager and reader
- Seahorse metabolic bioanalyser
- LiCOR Odyssey CLx infra-red detection system including InCell western module
- Zeptosens RPPA reader
- GeSim nanoplotter
- LiCONICs 220 stacking tower incubator
- EVOS Fl, bench top fluorescent microscope
- barcode printer; all instruments are barcode-read enabled
- dedicated and fully backed up server for data and image storage
Access and contact information
Each screen is unique and the VCFG team encourages researchers to work in the most biologically appropriate cell line and to think broadly in the scope of their analysis. Once data has been captured, it is always possible to re-analyse for additional information.
The VCFG team works with researchers each step of the way, through assay development, optimisation, transfection and bioinformatics analysis. This partnership begins by working with the laboratory head to discuss the screen and all related the logistics, before moving into the lab, obtaining reagents, being trained on the appropriate instruments and ultimately working with our dedicated bioinformatician to analyse your data. A comprehensive user guide and associated instrument guides are available to describe the different stages of a screen.
The nature of screening and assay development dictates screen slots cannot be locked in until an assay has passed rigorous statistical criteria. The VCFG team work with different users to schedule screens appropriately depending on downstream assay needs, offering a substantial number of screen slots a week.
All data generated remains the intellectual property of the screener.
A/Prof Kaylene Simpson - Head
Dr Iva Nikolic - Senior Research Officer, 2iC, CRISPR R&D
Jennii Luu - Lab manager, automation specialist
Karla Cowley - High content microscopy
Arthi Macpherson - Reverse Phase Protein Array platform
Robert Vary - Screening, lab support
Lucy Liu - Bioinformatics analyst
The VCFG is funded by:
- the Australian Cancer Research Foundation (ACRF)
- the University of Melbourne, Melbourne Collaborative Research Infrastructure Program
- the Australian Phenomics Network (APN) through the National Collaborative Research Infrastructure Scheme
- the Victorian Department of Economic Development, Jobs, Transport and Resources
- the NHMRC
- Cancer Council Victoria
- the Jack Brockhoff Foundation
- the Peter MacCallum Cancer Foundation.
VCFG Publications and Resources
For a VCFG publications list and other resources, please visit our publications and resources page.