Virus Screening Facility
The MRC WIMM Virus Screening Facility (VSF) provides a single point of call for lentivirus production and to facilitate lentiviral library creation aimed primarily at CRISPR/Cas9-based screening.
We provide assistance in all aspects of lentiviral screening, from initial experimental design through to library design and validation. We can help in virus production and purification, setting up of suitable transduction protocols, and offer help in data retrieval and the initial steps of data analysis.
CRISPR/Cas9 screening is becoming a widely used tool to identify novel gene targets in a wide variety of model systems. The technique focuses on the use of a gRNA/Cas9 complex either for the creation of indels mediated by the nuclease activity of Cas9 or transcription modulation using CRISPRa/CRISPRi.
A simple knockout screen can reveal sgRNAs that are enriched after treatment due to the selective advantage gained through the loss of a gene, while a negative screening approach uncovers gRNAs that are depleted due to selective disadvantage.
Alongside screening the VSF offers reagents and production advice, as well as basic lentivirus production. We have a library of lentiviral plasmids available, including a variety of promoters, selection markers and cloning strategies. Varieties of envelope and helper plasmids are also available. As standard, all lentiviral preparations will be pseudotyped with VSV-G to enable robust transduction of a wide variety of cell types, but a number of other pseudotyping vectors are available. We can produce lentiviral preparations at scales from 2 mL up to 500 mL, provided as supernatant from the production process or concentrated/purified in a number of ways.
We also provide: Crude cell supernatant, Membrane based centrifugation concentrators for up to 20 fold concentration and/or buffer exchange, Ultracentrifugation for 50-100 fold concertation, Chromatography purification.
Using our facility
The cost of reasonable consumables required for screening work will be covered for applicants from the MRC WIMM, Department of Oncology and those funded by the Oxford Biomedical Research Centre. Routine lentiviral production is available for internal applicants and will be charged to cover consumables and equipment maintenance.
Enquiries from external collaborators are encouraged; requirements and costings will be discussed on a per project basis.
Practical Considerations for Using Pooled Lentiviral CRISPR Libraries
McDade J.R. et al, (2016), Curr. Protoc. Mol. Biol, 115513, 311 – 31
Identification and characterization of essential genes in the human genome
Wang T. et al, (2015), Science, 350, 1096 – 101
Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis
Chen S. et al, (2015), Cell, 160, 1246 – 1260
A Genome-wide CRISPR Screen in Primary Immune Cells to Dissect Regulatory Networks
Parnas O. et al, (2015), Cell, 162, 675 – 686
Increasing the performance of pooled CRISPR–Cas9 drop-out screening
Cross B.C.S. et al, (2016), Scientific Reports, 6, 31782