Human CRISPR Knockout Pooled Library (GeCKO v2)
(Pooled Library #1000000048, #1000000049)
The human GeCKO (Genome-Scale CRISPR Knock-Out) lentiviral pooled libraries target early consecutive exons for genome editing.
- Depositing Labs
Controls1000 per half-library
This library will be delivered as two pooled DNA half-libraries in microcentrifuge tubes on blue ice (tubes labeled A and B). The tube's contents will not necessarily be frozen. For best results, minimize freeze-thaws.
For each half-library tube, you will receive:
For the 2 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. This plasmid is SHIPPED SEPARATELY at ambient temperature.
- 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)
Terms and Licenses
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 1000 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 2 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 (lentiCRISPRv1/2 and lentiGuide-Puro).
For additional information, visit Neville Sanjana Lab's Library screening resources website:
These pooled libraries were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the pooled libraries were described, and include Addgene in the Materials and Methods of your future publications.
Example for your Materials & Methods section:Human GeCKOv2 CRISPR knockout pooled library was a gift from Feng Zhang (Addgene # )
For your References section: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