Validated CRISPR site, which serves as an experimental control for the Cas9-D10A Nickase system. A three component positive control system consisting of a CMV-driven Cas9-D10A Nickase plasmid, a U6-driven guide RNA plasmid targeting the sense strand of the human EMX1 gene and a U6-driven guide RNA plasmid targeting the antisense strand of the human EMX1 gene.
Application
Functional Genomics/Target Validation
Creation 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
Serves as an experimental control for the CRISPR editing workflow using Cas9-D10A Nickase. Allows for validation of your system with the CRSIPR/Cas9 system. A positive result in a miss-match detection assay will indicate validation of your system.
Components
1 vial containing 1ug of a plasmid expressing the forward strand guide sequence. 1 vial containing 1ug of a plasmid expressing the reverse strand guide sequence. 1 vial containing 1ug of a plasmid expressing Cas9-D10A.
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 plasmid expressing a guide sequence to human EMX1 supplied at a concentration of 20ng/ul in 50ul. Sigma Cas9-D10A Nickase plasmid 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-prepping plasmids using endotoxin-free DNA purification kits prior to transfection.
Other Notes
Typical transfection concentrations used in literature are in the ranges of >= 1.0ug/UL and <= 5uL of Cas9-D10A Nickase plasmids combined with >= 1.0ug/UL and <= 5uL of U6-gRNA plasmids. (All dosages above assume 0.5 to 1 million cells nucleofected)
Learn about CRISPR Cas9, what it is and how it works. CRISPR is a new, affordable genome editing tool enabling access to genome editing for all.
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