Technology / Libraries

siRNA screening libraries

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RNA interference uses an RNA-guided RNA nuclease strategy to degrade specifically gene coding mRNA promoting transient (siRNA) or stable gene silencing (shRNA).

 

 

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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.

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Organization of the Druggable genome subset

 

Regarding siRNA subset libraries, the facility have a customized mouse and its corresponding human subset of 1,200 SiGenome SmartPool siRNAs targeting nuclear epigenetic chromatin remodelers (An RNAi screen of chromatin proteins identifies Tip60-p400 as a regulator of embryonic stem cell identity. Fazzio TG, Huff JT, Panning B. Cell 2008 Jul 11;134(1):162-74. doi: 10.1016/j.cel.2008.05.031)

Given the low amount of siRNA libraries, no cherry-picking or reformatting will be performed on these ones. However, pre-existing subsets (kinome, phosphatome, GPCRs, druggable genome, ...) can be used for screening purposes. 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 1. the human and mouse GeCKO v2 pooled CRISPR/Cas9 whole genome libraries (LentiCRISPRv2 vector) and 2. the human Brunello and mouse Brie GPP pooled CRISPR whole genome sgRNA libraries (LentiGuide vector). We also have the two Brunello kinome GPP sgRNA libraries (1-4 and 5-8)**. 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 GenomEast Microarrays and Deep Sequencing facility of the IGBMC. 

*Improved vectors and genome-wide libraries for CRISPR screening . Sanjana NE, Shalem O, Zhang F. Nat Methods. 2014 Aug;11(8):783-4. doi: 10.1038/nmeth.3047. 10.1038/nmeth.3047PubMed 25075903

**Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9 . Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, Smith I, Tothova Z, Wilen C, Orchard R, Virgin HW, Listgarten J, Root DE. Nat Biotechnol. 2016 Jan 18. doi: 10.1038/nbt.3437. 10.1038/nbt.3437 PubMed 26780180

 

Chemical compound libraries 

The Prestwick FDA-approved drug library (1,280 drugs), the SelleckChem FDA-approved library (1,430 drugs), the SelleckChem Epigenetic compound library (181 molecules) have 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. Other small, medium and large size small molecule libraries including the French National Chemolibrary or custom libraries can be screened at the facility.

Last update on Tue 27 Feb 2018

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