Virus Screening Facility

The Virus Screening Facility (VSF) has been set up to establish a single point of call for lentivirus production/advice and to facilitate lentiviral library creation, primarily for CRISPR screening.

The aim of the facility is to provide assistance in all aspects of lentiviral screening from initial experimental design through to virus production, purification, transduction and data retrieval/analysis.

Lentivirus Background

Lentivirus are a sub group of retrovirus that can stably insert a DNA sequence into the genome of a host. The main advantages of lentivirus compared to other viral systems are the ability to infect non-diving cells, large DNA packaging capacity (up to 10kb) and ease of production.

HIVvirus_structure
Figure1. HIV virus structure: The viral capsid contains two copies of the RNA viral genome bound to nucleocapsid proteins including the reverse transcriptase, protease, ribonuclease and integrase enzymes required for DNA production and insertion.

The wild type HIV virus includes 9 genes, 5 of which are required for production, regulation and integration (see table below). The remaining 4 are involved in viral replication/pathogenesis and are removed in lentiviral vectors.   

Gene

Function

gag

Core structural proteins (p24, SP1, NC, SP2, p6)

pol

Polymerase, transcribes RNA genome into DNA

env

Viral envelope gene, cleaved into gp120 and gp41

tat

Trans-activator of transcription, increases dsDNA generation (2nd generation vectors only)

rev

Transactivating protein involved in the export of unspliced viral mRNA from the nucleus

Table 1: Core genes required for lentiviral production.

Lentiviral vectors are classified as 2nd or 3rd generation depending on their requirement for tat and the separation of gag-pol and rev on two separate plasmids. The removal of tat dependency and an increased number of vectors for 3rd generation production reduces the chance of replication competent particles being produced.

Both generations of vector are Self INactivating (SIN) where a section of the 3’LTR is truncated. Upon transcription and integration the truncated 3’ LTR is transferred to the 5’LTR removing the transcription binding sites that would normally allow for production of the full-length viral genome. Therefore, only transcripts driven by internal promoters can be expressed. See figure 2 for the general structure of the integrated elements of a 3rd generation lentiviral transfer plasmid.

Advanced_viral_vector
Figure 2: A summary diagram of the integrated components of a third generation lentiviral transfer vector, for a detailed explanation of each component please see: Sakuma, T., Barry, M.A. & Ikeda, Y., 2012. Lentiviral vectors: basic to translational. Biochem J, 443(3)

For a more comprehensive background to lentivirus generations and plasmids please visit the viral vectors repository site at Addgene.

Multiplicity Of Infection (MOI) is the ratio of infectious particles to targets in an experiment and this is an important consideration when planning any experiment. An MOI of 1 indicates an average of one virus per cell, however MOI is not an absolute measure but a probability that follows Poisson distribution (do not be scared of the formula):

formula

This equation gives the probability (P) that a target cell will be infected by n viral particles at a given MOI (m). For example, the probability of a cell undergoing a single infection event after treatment with an MOI of 1 is 36.79% while 18.4% of cells will have two infections. This is visualised in figure 3.

This calculation is especially important when performing any kind of screening work, as cells infected at too high an MOI will undergo a significant rate of multiple infection events. In bulk screening methods, this would prevent the discrimination of enrichment scores returned from cells containing multiple gRNA/shRNA integrations. Doing single cell analyses as readout compensates, as long as the different barcodes can be discriminated from each other.

poisson_distibution
Figure 3: The Poisson distribution of viral infection by MOI. The percentage of a given cellular population plotted against the number of infections per cell at given MOI’s from 0.5 to 10.

Services Available

(1) Reagents and production advice

If you wish to discuss your own lentiviral preparations, protocols or experimental design please contact the VSF.

The VSF has a library of lentiviral plasmids available; these include 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. A number of other pseudotyping vectors are available. If your model system requires a different pseudotype please contact the VSF.

(2) Lentivirus production:

The VSF can produce lentiviral preparations at scales from 2 mL up to 500 mL. These can be provided as supernatant from the production process or concentrated/purified in a number of ways. We charge virus production to cover our consumables and running costs. Everyone is welcome to use the service within reasonable use. We offer:

Please contact the VSF to discuss project specific requirements and costings. Titration of vectors can be performed as long as a selection marker (FP expression or presence of a selection antibiotic marker) is present.

The virus production request form can be found here.

(3) CRISPR and shRNA Screening

CRISPR/Cas9 screening is becoming an increasingly popular tool to identify targets of interest in a number of model systems. Experimental conditions can allow for either a positive or a negative screen. A positive screen reveals sgRNA’s that are enriched after treatment (selective advantage) when compared to a control population while negative screens are used to look for gRNAs that are depleted (selective disadvantage). Figure 4 gives an overview about the screening pipeline. Any individual screen will be discussed in detail before starting the project, and input from both, the VSF and the Genome Engineering Facility is available.

Library complexity is entirely dependent on the experimental scope and design. A number of genome wide libraries for mouse and human cells are available, e.g. through Addgene as well as commercial providers such as the SIGMA (Mission shRNA), Dharmacon, Cellecta and ThermoFisher.

Many providers also offer defined shRNA and sgRNA libraries against gene subsets such as Epigenetic Modifiers, Kinases and Protease associated genes. Custom libraries can be generated for smaller projects either internally or through a number of commercial sources. Please contact Genome Engineering/VSF to discuss the various possibilities.

When planning your screening experiment some key points to consider are:

Funding bodies are providing support to cover working hours and general consumables, costs such as NGS sequencing or excess consumables will have to be met by the PI. The VSF committee will review all project proposals in strict confidence. The review process will focus primarily practical feasibility. The screening request form can be found here.

flowchart
Figure 4. An experimental flowchart for using pooled lentiviral CRISPR libraries in a Cas9 expressing cell line. McDade, J.R. et al., 2016. Practical Considerations for Using Pooled Lentiviral CRISPR Libraries. Curr. Protoc. Mol. Biol, 115513(5), pp.311–31.

 

If you would like to read more about the background and experimental considerations, the following publications are recommended:

McDade, J.R. et al., 2016. Practical Considerations for Using Pooled Lentiviral CRISPR Libraries THE BASICS OF CRISPR/CAS9 IN GENOME ENGINEERING. Curr. Protoc. Mol. Biol, 115513(5), pp.311–31.

Wang, T. et al., 2015. Identification and characterization of essential genes in the human genome. Science (New York, N.Y.), 350(6264), pp.1096–101.

Chen, S. et al., 2015. Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis. Cell, 160(6), pp.1246–1260.

Parnas, O. et al., 2015. A Genome-wide CRISPR Screen in Primary Immune Cells to Dissect Regulatory Networks. Cell, 162(3), pp.675–686.

Cross, B.C.S. et al., 2016. Increasing the performance of pooled CRISPR–Cas9 drop-out screening. Scientific Reports, 6(August), p.31782.