Application

Functional Genomics/Screening /Target Validation

Features and Benefits

Use CRISPR nucleases to knockout protein-coding genes to assess their function Efficiently screen the whole human genome (16,000+ genes) at the bench-top without robotics or specialized equipment Numerous built-in enrichment and depletion controls allow researchers to confidently gauge the success of their pooled screening experiments Lentiviral CRISPRs can infect a broad variety of mammalian cells by co-expressing a mammalian codon-optimized Cas9 nuclease along with a single guide RNA (sgRNA) to facilitate gene knockout. Use the one vector system for the human GeCKO version 2 libraries in applications where the Cas9 cannot be introduced into human cells (like in primary cells). Use puromycin to select after transduction.

General description

Developed by Feng Zhang′s lab at the Broad Institute, the human GeCKO v2 (all-in-one vector) libraries consist of over 100,000 unique gRNAs for gene knock-out in the human genome. Using an all-in-one lenti CRISPR vector which co-expresses Cas9 , gRNA with a puromycin selection marker, the version 2 vector produces nearly 10X higher titer virus than version 1. The human single vector system GeCKO pool is provided in 8 x 25 ul aliquots at a minimum titer of 5X10^8 TU/ml (measured by a p24 assay). The GeCKO human library is delivered as two half-libraries (A and B). It is recommended to screen both A and B libraries together, which will include 6 sgRNAs per gene (3 sgRNAs in each library). Both libraries contain 1000 non-targeting control sgRNAs. The "A" library also targets miRNAs (4 sgRNAs per miRNA).

Legal Information

All-in-One is a trademark of Sigma-Aldrich Co. LLC

CRISPR Use License AgreementLentiviral License Agreement

Other Notes

This product is for R&D use only, not for drug, household, or other uses. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices. Though the lentiviral transduction particles produced are replication incompetent, it is recommended that they be treated as Risk Group Level 2 (RGL-2) organisms in laboratory handling. Follow all published RGL-2 guidelines for laboratory handling and waste decontamination.

Preparation Note

Puro Kill Curve and Determining CFU (Colony Formation Unit) per mL. Prior to performing a library-scale screening, two preliminary experiments must be conducted. Visit Sigma.com/pooledscreening.