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GeCKO libraries for genome-scale CRISPR knock-out

GeCKO libraries for genome-scale CRISPR knock-out

The genome-scale CRISPR knock-out (GeCKO) v2 libraries enable rapid screening for loss-of-function mutations that produce a desired phenotype in human or mouse cells. GenScript is pleased to offer all eight GeCKO v2 CRISPR libraries developed in Feng Zhang's laboratory through a license with the Broad Institute.* GeCKO libraries are mixed pools of guide RNA targeting each gene and miRNA within the mouse or human genome; the improved GeCKO v2.0 libraries also contain non-targeting gRNA to serve as controls and utilize improved lentiviral vectors for high-titer lentivirus production and highly efficient transduction of primary cells. GeCKO libraries have been used in primary mouse and human cells, stem cells, cancer cells, and stable cell lines. GeCKO library screens allows you to identify genes that are essential for cell viability under different conditions, as well as genes whose loss enables drug resistance in cancer cells. For complete details of GeCKOv2 library design & construction, please see: Sanjana N.E., Shalem O., Zhang F. Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods. 2014 Aug;11(8):783-4. doi: 10.1038/nmeth.3047.

How to use GeCKO libraries

GeCKO libraries are a mixed pool of CRISPR guide RNAs that target every gene and miRNA in the genome. Each gRNA is cloned into a lentiviral vector optimized to produce high-titer virus for efficient lentiviral transduction of primary cells or cultured cell lines. A cell population should be transduced with the GeCKO library pool at a low MOI ensuring no more than 1 gRNA enters any given cell. After transduction, deep sequencing with NGS should be performed to assess gRNA representation in the cell pool before beginning a screening protocol. At the end of the screen, a second round of NGS is performed, and data analysis should be performed to identify the guides that were lost or enriched over the course of the screen. In order to identify true positive hits from a GeCKO library screen, you should identify genes for which multiple guides were enriched. A more detailed GeCKO screening protocol may be found on the Genome Engineering website.

GeCKO v2.0 Library Specifications

GeCKO libraries were designed to contain 6 single guide RNA (sgRNA) molecules targeting each genes within the human or mouse genome, as well as 4 sgRNA targeting each miRNA, and 1000 control (non-targeting) sgRNAs. The gRNA sequences are distributed over three or four constitutively expressed exons for each gene and were selected to minimize off-target genome modification. Each library was divided into two sublibraries, A and B, each containing 3 unique sgRNA for each gene; only library A contains 4 sgRNA targeting each of 1,864 miRNAs; both A and B contain the same 1,000 nontargeting control sgRNAs. The use of a single sublibrary maintains comprehensive genome-scale coverage but reduces the number of cells required to perform a screen, which is useful when cell numbers are limiting (for example, with primary cells or in vivo screens); alternatively, larger screens can be performed by combining both sublibraries.


GeCKO library specifications – guide RNA for human and mouse GeCKO sublibraries

All GeCKO library sequences are available in .csv files linked below:

GeCKOv2 Library Vector Options

To deliver a GeCKO library to cells via lentiviral vector with high efficiency, maximal viral titers have been observed when using a dual-vector delivery system, in which S. pyogenes Cas9 (lentiCas9-Blast) and sgRNA(lentiGuide-Puro) are delivered using separate viral vectors with distinct antibiotic selection markers. Alternatively, the library may be delivered in an all-in-one vector lentiCRISPRv2 which shows 10-fold higher viral titers than the original lentiCRISPR vector.

GenScript offers your choice of the following lentiviral vectors optimized for use with GeCKO library screens:

Vector Name
gRNA promoter
All-in-one vector?
Selection/Screening Markers
Vector Map

pLentiCRISPR v2
U6
Yes
Cas9 is driven by EFS promoter, with C-terminal DYK
AmpR, PuroR, BleoR
pLentiGuide-Puro
U6
No
pLentiCas9-Blast is provided free of charge for co-expression
AmpR, PuroR

Related Services for CRISPR/Cas9 genome editing

How to order GeCKO Libraries

Please inquire by emailing gene@genscript.com. GenScript generates amplified and validated GeCKO libraries according to the GeCKO library amplification protocol provided by the Feng Zhang laboratory, followed by next-generation sequencing to quantify guide RNA representation and pool complexity.

Legal Statement of GenCRISPR Services and Products (Updated on July 28, 2015):

  1. GenCRISPR™ services and products are covered under US 8,697,359, US 8,771,945, US 8,795,965, US 8,865,406, US 8,871,445, US 8,889,356, US 8,889,418, US 8,895,308, US 8,906,616 and foreign equivalents and licensed from Broad Institute, Inc. Cambridge, Massachusetts.
  2. .The products and the reagents generated from these services shall be used as tools for research purposes, and shall exclude (a) any human or clinical use, including, without limitation, any administration into humans or any diagnostic or prognostic use, (b) any human germline modification, including modifying the DNA of human embryos or human reproductive cells, (c) any in vivo veterinary or livestock use, or (d) the manufacture, distribution, importation, exportation, transportation, sale, offer for sale, marketing, promotion or other exploitation or use of, or as, a testing service, therapeutic or diagnostic for humans or animals.
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