Application

CMV-CAS9-2A-GFP plasmid has been used to induce additional sex combs-like1 mutations in human U937 leukemic cells. It has also been used in CRISPR/Cas9 analysis.CMV-CAS9-2A-GFP plasmid is suitable for functional genomics/target validation for:Creation of gene knockouts in multiple cell lines Complete knockout of genes not amenable to RNAiCreation of knock-in cell lines with promoters, fusion tags, or reporters integrated into endogenous genes

Components

1 vial containing 1 µg of Cas9-2A-GFP 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. The addition of a fluorophore that is translationally co-expressed with the Cas9 nuclease allows for easy visualization of successful transfection.

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 µg/µL and ﹤= 5 µL of Cas9 plasmid combined with >= 1.0 µg/µL and ﹤= 5 µL of U6-gRNA plasmids. (All dosages above assume 0.5 to 1 million cells nucleofected).

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.