Technology / Libraries
siRNA screening libraries
RNA interference uses an RNA-guided RNA nuclease strategy to degrade specifically gene coding mRNA promoting transient (siRNA) or stable gene silencing (shRNA).
The High Throughput Cell-based Screening Facility has acquired the Thermofisher Dharmacon On Target Plus SmartPool siRNA library targeting the human genome and the SiGenome SmartPool siRNA library targeting the mouse genome.
Regarding siRNA subset library, the facility have a mouse and its corresponding human subset of 1,200 SiGenome SmartPool siRNAs targeting nuclear epigenetic chromatin remodelers
The Qiagen Druggable genome library is also available as pooled siRNAs (4 sequences) as well as the Ambion SilencerSelect siRNA subset targeting the human receptome (1,450 genes). Kinome, phosphatome, GPCRs Subsets from this library can be used for screening purpose
Given the low amount of the genome libraries, no cherry-picking or reformatting will be performed on these ones. If you are interested in a particular subset not mentioned above, you can provide your own library.
CRISPR/Cas9 screening libraries
CRISPR/Cas9 is a recent genetic screening strategy that consists in a DNA nuclease enzyme Cas9 being targeted specifically to precise genomic regions by a bound RNA molecule (complementary sequence). This latter can be easily programmed by changing the sequence of 18-20 nucleotides ahead of a PAM protospacer adjacent motif (NGG in the cas of Streptococcus pyogenes). Cas9 recognizes the PAM and cleaves the genomic DNA generating double-strand breaks that are resolved by Non-Homologous End Joining (NHEJ) making frameshift insertion/deletion (Indel) and resulting in a loss-of-function allele. This strategy has been adapted to generate large-scale pooled libraries covering the human and mouse genome. We have acquired the human and mouse GeCKO v2 pooled CRISPR/Cas9 libraries (LentiCRISPR vector) developed in the laboratory of Feng Zhang*. Amplification and lentiviral packaging of the libraries are currently performed to allow positive and negative selection-based screening. Deep sequencing for hit identification can be performed at the Microarrays and Deep Sequencing facility of the IGBMC.
CRISPRi relies on the generation of catalytically inactive Cas9 by introducing point mutations in the two catalytic residues (D10A and H840A). Inactive dCas9 is then unable to cleave dsDNA but retains the ability to target specifically DNA using sgRNA. Together sgRNA and dCas9 are the two key components of a powerful system for gene-specific regulation in any organism. WE can turn out dCAS9 into a transcriptional repressor by fusing the Krüppel associated box (KRAB) domain or into a transcriptional activator by fusing or recruiting VP16 modules instead. Pooled lentiviral CRISPRi and CRISPRa Libraries targeting the human genome have been developed in the laboratory of Jonhathan Weissman** and are available at Addgene.
*Improved vectors and genome-wide libraries for CRISPR screening. Sanjana, N., Shalem, O., Zhang, F. Nature Methods. Nat Methods. 2014 Aug;11(8):783-4.
**Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Gilbert LA, Horlbeck MA, Adamson B, Villalta JE, Chen Y, Whitehead EH, Guimaraes C, Panning B, Ploegh HL, Bassik MC, Qi LS, Kampmann M, Weissman JS.Cell. 2014 Oct 23;159(3):647-61
If interested by this technology, you can contact us to discuss about your project.
Chemical compound libraries
The Prestwick FDA-approved drug library (1,280 drugs) has been acquired and can be screened at the facility. It provides an easy pilote before large scale chemical screening and is also an interesting library for repositioning drugs accelerating their transfer to clinical applications