Human CRISPR Knockout Pooled Library (GeCKO v2)
(Pooled Libraries #1000000048, #1000000049)

• Purpose:

  The human GeCKO (Genome-Scale CRISPR Knockout) lentiviral pooled libraries target early consecutive exons for genome editing.

• Vector Backbone:

  • lentiCRISPR v2 (Plasmid #52961) backbone (one-vector system) - expresses Cas9
  • lentiGuide-Puro (Plasmid #52963) backbone (two-vector system) - does not express Cas9

• Depositing Labs: Feng Zhang
• Publication: Sanjana et al Nat Methods. 2014 Aug;11(8):783-4. doi: 10.1038/nmeth.3047.

Library Details

• Species: Human
• Genes targeted: 19,050
• gRNAs: 123,411
• Controls: 1,000 per half-library
• 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.

This library will be delivered as two pooled DNA half-libraries in microcentrifuge tubes labeled A and B.

• Volume: ∼20 µL
• Concentration: 50 ng/µL

For the two-vector system ONLY: You will also receive a bacterial stab of the lentiCas9-Blast plasmid (Addgene #52962). Please do NOT order this plasmid in addition to this library.

Resource Information

• Protocols:
  • For the most up-to-date and detailed instructions on using this library, please see the protocol paper from Joung, et al.
  • Library Amplification Protocol (269.2 KB)
  • gRNA cloning in lentiCRISPRv1, lentiCRISPRv2 and lentiGuide-Puro vectors (2.3 MB)
• Depositor Data:
  • FASTQ files for GeCKO libraries (589 MB)
  • Human library A gRNA sequences (2.8 MB)
  • Human library B gRNA sequences (2.4 MB)

Terms and Licenses

Academic/Nonprofit Terms:

• UBMTA

Industry Terms:

• Not Available to Industry

Trademarks:

• Zeocin® is an InvivoGen trademark.

Depositor Comments

In the library amplification protocol, we describe how to amplify GeCKO v2.0 DNA plasmids to have sufficient quantity to produce lentivirus, while maintaining full library representation. The GeCKO v2 libraries consist of over 100,000 unique gRNAs for gene knock-out in either the human or mouse genome.

Each species-specific library is delivered as two half-libraries (A and B). When used together, the A and B libraries contain 6 sgRNAs per gene (3 sgRNAs in each library). We recommend screening the entire library (A and B) when possible but if adequate representation cannot be obtained with the entire library, screening can be performed with one half-library. Since more cells are needed in the screen as number of constructs in the library increases, this design has the flexibility of screening with just the A library (3 sgRNAs per gene) at a smaller scale or screening the full library (6 sgRNAs per gene).

Both A and B libraries contain 1,000 control sgRNAs designed not to target in the genome. The A library also targets miRNAs (4 sgRNAs per miRNA). Complete sequences and targets of all sgRNAs in the A and B libraries are available in CSV format above.

The two-vector format with the library in lentiGuide-Puro has the advantage of higher titer for the library virus but requires cells to already contain Cas9 (usually genomically integrated using lentiCas9-Blast). There is also a protocol below for cloning custom gRNA sequences of your own design into any of the vectors (lentiCRISPR v1/2 and lentiGuide-Puro).

For additional information, visit Neville Sanjana Lab's Library screening resources website:
http://sanjanalab.org/lib.html