Victorian Centre for Functional Genomics

The Victorian Centre for Functional Genomics (VCFG) at Peter Mac offers biomedical researchers Australia-wide the ability to perform novel discovery-based functional interrogation all genes in the genome, or selected boutique collections using multiple platforms including CRISPR/cas9, small interfering RNA (siRNA), micro RNA (miRNA) and long non-coding RNA (lncRNA) and short hairpin RNA (shRNA).

The VCFG facilitates small scale drug screens using commercially available compounds or your own source. The VCFG has established a 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 VCFG also offers Short Tandem Repeat (STR) profiling for cell line authentication.

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, headed by A/Prof Kaylene Simpson.

Platforms

CRISPR/Cas9 screening

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. 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.

siRNA platform

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.

miRNA platform

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.

shRNA platform

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.

Reverse Phase Protein Array platform

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 100 antibodies to be screened (the collection is continually expanding).  Large or small scale screens can be performed, with the capacity designed to dramatically reduce the need to run western blots.  Up to 70 lysates can be analysed per antibody, with the capacity for 6 antibodies per glass slide.  As with all platforms, the VCFG has a bioinformatics pipeline for quantitative analysis of expression levels.  For more details contact A/Prof Kaylene Simpson.

Resources

Available resources

Pooled screening

  • human whole-genome CRISPR pooled library (Brunello)
  • mouse whole-genome CRISPR pooled library (Brie)
  • individual shRNA and sgRNA constructs for assay development
  • human whole genome version 3 shRNAmiR 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

Arrayed screening

  • custom synthetic crRNA libraries
  • 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
  • wound healing assay
  • access to MetaCore (GeneGO) and Oncomine premium analysis tools.

Quantitative Proteomics

  • Zeptosens lysis buffer
  • Over 100 antibodies (primarily directed to human target genes raised in rabbit but with mouse reactivity)
  • Capacity for western blotting validation using the LiCOR Odyssey and accessing the antibody collection.

Available instrumentation

  • high-content microscopy using fully automated Cellomics Arrayscan VTi and Cell Insight (x2)
  • Caliper ALH3000 liquid handling robot for all transfection and speciality liquid handling applications
  • BioTek 406: three units that aspirate and dispense media, cells, fix and stain and add drugs
  • Cytation3, a sophisticated plate imager and reader
  • Synergy H4 high-throughput plate 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.

Facility staff

Head: A/Prof Kaylene Simpson

Senior Research Officer, 2IC: Dr Iva Nikolic

Bioinformatics Analyst: Dr Piyush Madhamshettiwar

Cell Biology Specialist: Jennii Luu

Research Assistant VCFG: Karla Cowley

Research Assistant RPPA: Arthi Macpherson

The VCFG team. Front left to right: Arthi Macpherson, Jennii Luu, Piyush Madhamshettiwar, Kaylene Simpson. Back left to right: Daniel Thomas, Iva Nikolic, Karla Cowley.

Funding acknowledgEment

The VCFG is funded by:

VCFG Publications and Resources

For a VCFG publications list and other resources, please visit our publications and resources page.