CRISPR UNIVERSAL NEGATIVE CONTROL 1

Description

• General description: 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.
• Application: Functional Genomics/Target ValidationCreation of gene knockouts in cell lines Creation of knock-in cell lines with promoters, fusion tags or reporters integrated into endogenous genes
• Features and Benefits: Allows for expression of Cas9 and GFP without specific targeting of the CRISPR/Cas9 system.
• Components: 1 vial containing 1ug of U6-gRNA/CMV-Cas9-GFP plasmid expressing a non-targeting guide sequence.
• Principle: 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.
• Physical form: Sigma U6-gRNA/CMV-Cas9-GFP plasmid expressing a non-targeting guide sequence supplied at a concentration of 20ng/ul in 50ul.
• Preparation Note: Sigma CRISPR plasmid products are delivered as mini-prep aliquots,which may not be suitable for transfection into particular cell types. For best results, we advise maxi-preppingplasmids using endotoxin-free DNA purification kits prior to transfection.
• Other Notes: 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)
• Legal Information: CRISPR Use License AgreementLentiviral and WPRE License Agreements