Two Vector CRISPR/Cas9 System The CRISPR/Cas9 system can be used for knocking out gene expression by using a combination of sgRNA and the Cas9 nuclease. The Two Vector CRISPR/Cas9 system allows for faster knockout of the target gene in cells by first selecting cells expressing high levels of Cas9. The Two Vector CRISPR/Cas9 system is also great for creating custom sgRNA libraries for screening assays. Expression of Cas9 and sgRNA from Separate Vectors to Optimize Knockouts • Obtain higher titers for Cas9 for difficult-to-transduce cells • Reduce noise in screening experiments by standardizing expression of Cas9 • Cas9 and sgRNA vectors contain different antibiotic resistance genes for easy selection • Tet-inducible and constitutive sgRNA expression available pRSG16-U6-sg-UbiC-TagRFP-2A-Puro 8.0 kb AmpR promoter Knock Out Targets with Lentiviral sgRNA Constructs • You provide the RefSeq number or gene ID • We design 3-5 sgRNA constructs that target the transcript 2-Vector vs. 1-Vector Titers Titer (TU/ml) 1.E+07 1.E+06 1.E+05 1.E+04 sgRNA vector Cas9 vector 2-Vector System (877) 938-3910 sgRNA/Cas9 vector pR-CMV-Cas9-2A-Hygro 11.6 kb When transducing many thousands of constructs into large populations of cells, as is required for loss of function screens with pooled sgRNA libraries, high titers are a necessity. The 2-Vector CRISPR system provides higher titers than the 1-vector system and allows selection of cells expressing a high level of Cas9 before introducing the sgRNA, which leads to more efficient knockouts with less variability for screens. 1-Vector System www.cellecta.com High Expression of Cas9 Increases Knockout Rate 120 % GFP+ Cells 100 80 60 40 20 0 Cas9 (1 copy/cell) Cas9 (3 copies/cell) Cas9 (1 copy/cell) + sgRNA (1 copy/cell) Cas9 (3 copies/cell) + sgRNA (1 copy/cell) Cells expressing GFP were transduced with Cas9 at high MOI producing a population with approximately 3 Cas9 per cell on average, or a low MOI generating a population of approximately 1 per cell on average. After selecting the Cas9 transductants with hygromycin, each population of cells was then transduced with the same sgRNA to GFP and grown for 9 more days in media containing puromycin. Cells with a higher number of integrated copies of Cas9 have 5-fold fewer GFP-positive cells. Related CRISPR Products & Services Human CRISPR Genome-Wide sgRNA Library Agilent Oligo Synthesis • Built using Agilent’s solid support oligonucleotide synthesis method • Provided as either plasmid or packaged pooled sgRNA library Custom CRISPR / sgRNA Libraries • You decide which genes you want to knock out • Cellecta designs 3-5 sgRNAs per gene (or to your specifications) sgRNA T6 barcode Oligo Pool PCR & Cloning Plasmid sgRNA Library Create Knockout Cell Lines with CRISPR • Design and cloning of sgRNA constructs • Viral packaging for sgRNA constructs and transduction into desired cell type • Confirmation by PCR that both alleles are out-of-frame • Functional assays such as proliferation, viability screens, or pathway activation assays available (877) 938-3910 Oligo Detachment 2 Lentivirus Production Packaged, Pooled sgRNA Library www.cellecta.com