Developed at the Broad Institute by Feng Zhang′s lab, the human GeCKO v2 (two vector system) libraries consist of over 100,000 unique gRNAs for gene knock-out in the human genome. Using a dual lenti CRISPR vector wherein the pooled libraries will only express the gRNA (with the lentiGuide-Puro vector), this system produces over 100X higher titer virus compared to version 1. The lentiGuide-Puro pool should be used only in cell lines with Cas9 already integrated (which can be generated using a separate lenti-Cas9-Blast vector). Sigma′s lentiviral human GeCKO pool guide RNA only is provided in 8 x 25 ul aliquots at a minimum titer of 5X10^8 TU/ml (measured by a p24 assay). Each species-specific 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).
Functional Genomics/Screening /Target Validation
Cechy i korzyści
- 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 transducing a single guide RNA (sgRNA) to a Cas9-expressing human cell line to facilitate gene knockout for screening applications.
- Use the dual vector system for the human GeCKO version 2 libraries for human cell lines that have Cas9 already integrated into the genome.
- Use puromycin gRNA selection after transduction.
Uwaga dotycząca przygotowania
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.
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.