CRISPR/Cas 9 Cell Screening Facility
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.
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.
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|
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.
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.