Application

Functional Genomics/Target Validation Creation of gene knockouts in multiple cell lines Complete knockout of genes not amenable to RNAi Creation of knock-in cell lines with promoters, fusion tags or reporters integrated into endogenous genes

Components

1 vial containing 1ug of Cas9 plasmid.Please note, product does not contain guideRNA sequence. This must be purchased separately through the Custom CRISPR product tab.

General description

The Cas9 expression plasmids use the CMV promoter for strong transient expression of Cas9. Alternate promoters can be substituted by replacement of CMV using MluI and NheI. Also, the Cas9 expression plasmids can be linearized using XbaI for T7-based mRNA production.

Legal Information

CRISPR Use License Agreement

Other Notes

Must be used in conjunction with a U6-gRNA plasmid in order to mediate a double strand break in the DNA. Typical transfection concentrations used in literature are in the ranges of >= 1.0 ug/uL and ﹤= 5 uL of Cas9 plasmid combined with >= 1.0 ug/uL and ﹤= 5 uL of U6-gRNA plasmids. (All dosages above assume 0.5 to 1 million cells nucleofected)

Physical form

Sigma Cas9 plasmid DNA is supplied at concentrations 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-prepping plasmids using endotoxin-free DNA purification kits prior to transfection.

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.