Gene Editing & Screening: Overview

Gene Editing & Screening
Overview
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Gene Editing & Screening: Overview

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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.