CRISPR/Cas 9 Cell Screening Facility

CRISPR Group

Introduction

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 or Cas9 derivatives to knockout, repress, or activate expression of target genes. Several pooled lentiviral CRISPR libraries have been developed and are now publicly available.  Here at the TDI we have Pooled Lentiviral CRISPR genome knockout and CRIPSR gain of function libraries.

 

Daniel Ebner, Elena Navarro Guerrero and Dylan Jones

 

Background

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes denoted as CRISPR/Cas9 system is a targeted gene-editing tool adapted from Streptococcus pyogenes that enables the permanent knockout of target genes. Single Guide RNAs (sgRNA) direct the Cas9 nuclease to a specific genomic region, upon which the Cas9 cleaves the target gene that is repaired by Non-Homologous End Joining (NHEJ) disrupting the open reading frame of the targeted gene by causing an InDel frameshift and/or premature stop codon. (Figure 1).
 
Figure 1.  Gene editing with Cas9.Figure 1. Gene editing with Cas9.

Knock-down and activation libraries

The CRISPR/Cas9 system is ideal for genome-wide knockout and gain of function screening experiments due to the ease of generating gRNAs and the efficiency and irreversibility of Cas9-mediated genetic modifications. Here at the TDI we have pooled lentivirus CRISPR knock-out and activation libraries that target all the genes in human and mouse genome.   
 
Table 1.  List of CRISPR library 
 
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
SAM v1 - 3 plasmid system Activation Human 3 70,290
SAM v1 - 3 plasmid system Activation Mouse 3 69,716
SAM v2 - 2 plasmid system Activation Human 3 70,290

 

Virus production 

We have the facility to produce lentivirus for pooled CRISPR lentivirus and provide advice (Figure 2). Most of the CRSPR libraries in table 1 have been used to make Lentivirus and is available for use in screenings. 

We also have Virus Like Particles(VLP) containing Vpx, which will help to increase lentivirus transduction.  Usefull in difficult to transduce cells like hematopoietic stem and progenitor cells.

 

Lentivirus

Figure 2. Schematic diagram of Lentivirus production

 

 

Screening and sequencing

The aim of the facility is to provide assistance in all aspects of lentiviral CRSPR screening from initial experimental design, virus production, purification, transduction, screening and  FACS sorting.   We can perform FACS sorting of the pooled genome-wide CRISPR screens using the benchtop SH800 cell sorter. SH800 has a 488nm excitation laser and permits sorting of a wide range of cell sizes and applications using different microfluidics sorting chips (70 μm, 100 μm, and 130 μm). 

For sequencing, we can help and assist in genomic DNA extraction and genomic PCR to prepare samples for sequencing. We have PCR primers for all of the CRISPR libraries and the technical resources and knowledge.