CRISPR UNIVERSAL NEGATIVE CONTROL 2

Description_x000D_ General description_x000D_ Universal negative control CRISPRs have been designed not to recognize any sequence in the human, mouse or rat genome. A single vector format is provided which includes the Cas9 expression cassette and gRNA sequence. This vector includes GFP which is co-expressed from the same mRNA as the Cas9 protein via a 2A peptide linkage and enables for tracking of transfection efficiency or enrichment in cell populations via FACS._x000D_ Application_x000D_ Functional Genomics/Target Validation_x000D_ • Creation of gene knockouts in cell lines_x000D_ • Creation of knock-in cell lines with promoters, fusion tags or reporters integrated into endogenous genes_x000D_ Features and Benefits_x000D_ Allows for expression of Cas9 and GFP without specific targeting of the CRISPR/Cas9 system._x000D_ Components_x000D_ 1 vial containing 1ug of U6-gRNA/CMV-Cas9-GFP plasmid expressing a non-targeting guide sequence._x000D_ Principle_x000D_ 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._x000D_ Physical form_x000D_ Sigma U6-gRNA/CMV-Cas9-GFP plasmid expressing a non-targeting guide sequence supplied at a concentration of 20ng/ul in 50ul._x000D_ Preparation Note_x000D_ Sigma CRISPR plasmid products are delivered as mini-prep aliquots,_x000D_ which may not be suitable for transfection into particular cell types. For best results, we advise maxi-prepping_x000D_ plasmids using endotoxin-free DNA purification kits prior to transfection._x000D_ Other Notes_x000D_ Typical transfection concentrations used in literature are in the ranges of 2.0 to 8.0ug of the single vector expressing the guideRNA and Cas9 . (All dosages above assume 0.5 to 1 million cells nucleofected)_x000D_ Legal Information_x000D_ CRISPR Use License Agreement_x000D_ Lentiviral and WPRE License Agreements