Gene Editing & Screening

Ralph Garippa (Core Head)

Office phone

646-888-3339

Office fax

646-888-3347

Email(s)

garippar@mskcc.org

The Gene Editing & Screening Core Facility (GES Core, known as the RNAi Core Facility from 2013 to 2017) provides MSKCC investigators with two RNA-guided technologies, RNA Interference (RNAi) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), to study individual genes, dissect signaling pathways, and identify potential drug targets. The GES Core provides the investigators with individual clone and pooled library of shRNA (RNAi) and sgRNA (CRISPR). The GES Core also conducts pooled library screening of shRNA and sgRNA by next generation sequencing, cell line production with gene knock-in and knock-out, and arrayed library screening of a human full-genome siRNA and various compound libraries in microtiter plate format.

Services

shRNA cloning - Individual clone and pooled library

  • RNA polymerase II-transcribed miR-E shRNA with high potency and specificity designed with the SpalshRNA algorithm.
  • Options including transduction (lentiviral or retroviral), expression (constitutive or inducible), fluorescence reporter (GFP, dsRED etc), and selection marker (puromycin, hygromycin etc).
  • Various target-focused pooled libraries including human and mouse druggable genome, epigenome, and kinome.
  • Additional services including virus packaging and transduction.

sgRNA cloning - Individual clone and pooled library

  • Options including fluorescence reporter (GFP, dsRED etc), and selection marker (puromycin, hygromycin etc).
  • Full-genome libraries including GeCKO v1 (human), GeCKO v2 (human and mouse), Brie (mouse), Brunello (human), and SAM (human).
  • Additional services including virus packaging and transduction.

Gene knock-in and knock-out cell line production

Pooled library screening - shRNA and sgRNA

  • Assay development including transduction optimization.
  • Screening execution, genomic DNA extraction, and PCR for next generation sequencing.
  • Data analysis.

Arrayed library screening - siRNA and compound

  • Human full-genome siRNA library (siGenome, Dharmacon).
  • Bioactives and FDA-approved drug libraries, target focused inhibitor libraries against kinase and GPCR, and diversity libraries.
  • Assay development and semi-automated screening in 384-well plate density format.
  • Cell viability assay and biochemical assay with EnSpire multimode plate reader (PerkinElmer).
  • High content cell imaging assay with IN Cell Analyzer 6000 laser-based confocal imaging platform (GE Healthcare) and Columbus image analysis system (PerkinElmer).

Pooled shRNA and sgRNA libraries ready off the shelf

Library #Gene #Subpool #shRNA/sgRNA per gene Control
shRNA human druggable genome 2,263 6 5 yes
shRNA human cancer metabolome 639 2 5 yes
shRNA human DNA damage repair 1,825 5 5 yes
shRNA mouse DNA damage repair 310 6 10 no
shRNA human kinome 526 3 5 yes
shRNA human epigenome 565 2 5 yes
shRNA human phosphatase 254 1 5 yes
shRNA human nuclear hormone receptor 49 1 5 yes
shRNA human oncogene (1) 62 1 6 yes
shRNA human tumor suppressor (1) 73 1 6 yes
shRNA mouse oncogene (1) 62 1 6 yes
shRNA mouse tumor suppressor (1) 73 1 6 yes
shRNA mouse Phagosome-proteosome 1,385 10 6 no
shRNA mouse drugged genome 446 3 5 yes
shRNA mouse DNA methylation 495 5 5 yes
sgRNA human SAM library (2) 23,430 1 3 yes
sgRNA human Brunello library (3) 19,114 1 4 yes
sgRNA mouse Brie library (3) 19,674 1 4 yes
sgRNA human epigenome 565 1 4 yes
sgRNA human druggable genome 2,263 6 4 yes
sgRNA human GeCKO library v2 (4) 19,050 2 6 yes
sgRNA mouse GeCKO library v2 (4) 20,611 2 6 yes
sgRNA human GeCKO library v1 (5) 18,080 1 2 to 4 yes

References:
1. Vogelstein et al., “Cancer Genome Landscapes” Science 2013, 339:1546-1558
2. Konermann et al., “Genome scale transcriptional activation by an engineered CRISPR-Cas9 complex” Nature 2015, 517:583-588.
3. Doench et al., “Optimized shRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9” Nat Biotechnol 2016, 34:184-191.
4. Sanjana et al., “Improved vectors and genome-wide libraries for CRISPR screening” Nature Methods 2014, 11:783-784.
5. Shalem et al., “Genome-scale CRISPR-Cas9 knockout screening in human cells” Science 2014, 343:84-87.