The Cas9-D10A nickase plasmid co-expressing RFP uses the CMV promoter for strong transient expression of Cas9-D10A. Alternate promoters can be substituted by replacement of CMV using MluI and NheI. Also, the Cas9D10A expression plasmids can be linearized using XbaI for T7-based mRNA production.
Application
Functional Genomics Use of Paired Cas9 Nickase + RFP for:
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-D10A Nickase-RFP plasmid.
Please note, this product does not contain any guide RNA sequence. A pair of gRNA plasmids must be purchased separately through the Custom CRISPR paired nickase product tab.
Physical form
2 ug of Sigma Cas9-D10A nickase-RFP plasmid
Other Notes
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. RFP is co-expressed from the same mRNA as the Cas9 nickase protein via a 2A peptide linkage, enabling tracking of transfection efficiency and enrichment of genome editing activity in cell populations via fluorescence activated cell sorting (FACS).
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