Human Genome-Wide CRISPR Knockout (LGR Backbone)
(Pooled Library #254659, #254660, #254661)

• Purpose:

  The Human Genome-Wide CRISPR Knockout (LGR Backbone) libraries may be used for pooled, genome-wide CRISPR screens in human cells expressing the correct CRISPR machinery. These libraries contain gRNAs for knockout (CRISPRko) of protein coding genes in the human genome for packaging and delivery by lentivirus. The three libraries are identical in sgRNA content and differ only in their fluorescent marker (BFP, eGFP, or mCherry), allowing users to select a variant compatible with reporters already in use in their cells or to run multiple libraries in parallel in the same experiment.

  These libraries use the sgRNA design reported by Doench et al. (2016) for Brunello (Addgene #73179 and #73178), but are delivered in different backbones.

• Vector Backbone: 254659 hCRISPRko-Brunello-LGR-Backbone-BFP:

  • pLGR_dual_guide-Puro-mycNLS with BFP (PDF, 250 KB; GB, 19 KB) - does not express Cas9

  254660 hCRISPRko-Brunello-LGR-Backbone-eGFP:

  • pLGR_dual_guide-Puro-mycNLS with eGFP (PDF, 277 KB; GB, 19 KB) - does not express Cas9

  254661 hCRISPRko-Brunello-LGR-Backbone-mCherry:

  • pLGR_dual_guide-Puro-mycNLS with mCherry (PDF, 246 KB; GB, 20 KB) - does not express Cas9

• Depositing Labs: Alexander Brown, Laboratory for Genomics Research (LGR)
• Publication: Laboratory for Genomics Research (LGR) - Human CRISPR Pooled Libraries

Library Details

• Species: Human
• Genes targeted: 20,367 loci
• gRNAs: 81,776
• Controls: 495 NTCs and 77 negative intron controls
• Lentiviral generation: 3rd

Library Shipping

Each library is delivered in a microcentrifuge tube on blue ice. The tube's contents will not necessarily be frozen. For best results, minimize freeze/thaws.

• Volume: ∼20 µL
• Concentration: 100 ng/µL

Resource Information

• Protocols:
  • Library Amplification Protocol (PDF, 463 KB)
  • PCR Protocols (DOCX, 20 KB)
• Depositor Data:
  • List of gRNA sequences (TSV, 3.2 MB)

Terms and Licenses

Academic/Nonprofit Terms:

• UBMTA
• Evrogen Limited Use Label License for FPs

Industry Terms:

• Not Available to Industry

Trademarks:

• Zeocin® is an InvivoGen trademark.

Depositor Comments

Growth issues were noticed when using pre-mixed media and LB plates. Because of this, we recommend using MegaX DH10B™ T1R Electrocomp™ Cells (Invitrogen #C6400-03) cultured at 37°C, which is what we use to generate all our libraries. Additionally, we recommend switching from ampicillin to carbenicillin to prevent satellite colonies.