CRISPR loss of function screening

Introduction

This overview is designed to be an introduction to CRISPR pooled screen at the Target Discovery Institute, Nuffield Department of Medicine, University of Oxford. Below you will find information detailing all aspects of CRISPR screening performed at our facility. The TDI benefits from a broad range of experience across a wide range of screens and our scientists have extensive knowledge of high throughput screening. We are available for consultation at the earliest stages of assay conception and we will work closely with each investigator through all stages of assay development, optimization and screening.

 

Background

CRISPR/Cas9 technology is ideally suited for genome-wide screening applications due to the ease of generating guide RNAs (gRNAs) and the versatility of Cas9 to knockout expression of target genes. Pooled lentiviral CRISPR libraries are a heterogenous population of lentiviral transfer vectors, each containing an individual gRNA targeting a single gene in a given genome.

 

Library description

Here at TDI we have employed a single CRISPR/Ca9 sgRNA lentiviral system called GeCKO vs2 (Genome-scale CRISPR Knock-Out).  This consists of pooled sgRNA librarary that were developed by the Zhang lab (Sanjana  et al., Nature Methods 2014) and and provided by Addgene (Figure 1)

Figure 2.  One vector GeCKO system
Figure 1. One vector GeCKO system

 

The GeCKO libraries consist of specific sgRNA sequences for gene knock-out in either the human or mouse genome. Each species-specific library is delivered as two half-libraries (A and B). When used together, the A and B libraries contain 6 sgRNAs per gene that target 19,050 genes in human and 20,611 in mouse (3 sgRNAs in each library, Table 1). Both A and B libraries contain 1000 control sgRNAs designed not to target in the genome. Libraries also contain sgRNAs that target miRNAs (4 sgRNAs per miRNA). 

Table 1
Table 1. GeCKO human and mouse library

All library sequences for screen readout are available for download.

Lentivirus for all libraries are produced at the TDI.

Optimisation

Several optimisation steps will have to be carried out with cells of choice for optimal screening

  1. Cell density and Growth kinetics
  2. Cell tolerance to Polybrene
  3. Kill curve with Puromycin
  4. Lentivirus functional titre to obtain multiplicity of infection (MOI) of 0.3

Pooled sgRNA Cas9 lentiviral library is transduced into target cells at MOI of 0.3, which are then selected for with puromycin, so that most cells receive only one copy of sgRNA, thus knocking out only one target gene per cell. The MOI is the ratio of viral particles to cells and represents the total number of viral particles that are added to a pool of cells. In pooled screening, it is crucially important that each cell in the pool is transduced with only one sgRNA construct to ensure the observed phenotype per cell is the result of one viral particle integration into the cellular DNA. However, by reducing the MOI the number of non-transduced cells increases.  Therefore the MOI has to be carefully decided in advance in order to keep the screen manageable. To avoid multiple integration, we recommend using a low MOI of 0.3-0.5, resulting in 25-39% positive transduced cells (Figure 2).

 

MOI
Figure 2. Effect of MOI on number of integrations per cell
 

Screening

For screening, cells will be transduced with the CRISPR Lentivirus using low MOI of 0.3-0.5 and coverage of 300-500.  Coverage represents the number of cells transduced with a single gRNA, so a coverage of 500 represents 500 cells transduced with a single gRNA.
 
Using a GeCKO library A with 65389 gRNA and coverage of 500, 32694500 cells will be transduced. In addition, with MOI of 0.3-0.5 and puromycin selection leading to 75% cell death, up to approximately 130 million cells will be needed for human library A transduction.
 
Cells will then be selected for 7 days with puromycin and gown to required amount for screening. For a typical dropout screen with a single drug, at least 33 million cells will be maintained and collected for day 0 reference, control arm and drug treated arm.  Length of screening will be based on the cell type and treatment, but typically cells will be collected between cell doubling 6 and 12 from start of screening.
 
Cells collected will be frozen and used for genomic extraction, PCR and submitted to the Wellcome Trust Centre Human Genetics for NexGen Sequencing (Figure 3).  The frequency of guide sequences will be read and targets with a statistically significant difference between conditions and reference control samples will be identified. 
plan
Figure 3. Screening outline

 

Available Knockout library in the TDI

The bellow pooled libraries are avilable as plasmids and Lentivirus in the TDI.  Please get in touch with Dylan Jones or Daniel Ebner for library request, advice or help in screening. 

Name

Library Type

Species

gRNAs per gene

Total gRNAs

 Human GeCKO v2

Knockout

Human

6

123,411

 Mouse GeCKO v2

Knockout

Mouse

6

130,209

 Toronto KnockOut vs1

Knockout

Human

12

176,500

 Toronto KnockOut vs3

Knockout

Human

4

70,948