Cas9 RFP Lentiviral Particles

Functional Genomics/Target Validation

• Creation of gene knockouts in multiple cell lines
• Complete knockout of genes not amenable to RNAi
• Creation of cell lines stably expressing Cas9-RFP

CRISPR/Cas systems are employed by bacteria and archaea as a defense against invading viruses and plasmids. Recently, the type II CRISPR/Cas system from the bacterium Streptococcus pyogenes has been engineered to function in eukaryotic systems using two molecular components: a single Cas9 protein and a non-coding guide RNA (gRNA). The Cas9 endonuclease can be programmed with a single gRNA, directing a DNA double-strand break (DSB) at a desired genomic location. Similar to DSBs induced by zinc finger nucleases (ZFNs), the cell then activates endogenous DNA repair processes, either non-homologous end joining (NHEJ) or homology-directed repair (HDR), to heal the targeted DSB.

Ready-to-use Cas9-RFP lentiviral particles enable immediate transduction of a wide range of cell lines. Cas9-RFP lentiviral particles efficiently and stably integrate Cas9 and RFP linked by a 2A peptide and driven by the EF1 alpha promoter (EF1a-Cas9-2A-GFP) for strong expression in both dividing and non-dividing cell lines. They are ideal for avoiding the tedious lentivirus production process and are one part of a two part CRISPR system with individual Cas9 and gRNA expression vectors. For easy fluorescence microscopy a MOI greater than 200 should be employed.

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VP/ml is the concentration unit of measure for viral titer estimated by p24 assay.