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

Components

1 vial containing 1ug of U6-gRNA plasmid expressing a guide sequence to human EMX1. 1 vial containing 1ug of Cas9 plasmid.

Features and Benefits

Serves as an experimental control for the CRISPR editing workflow using WT Cas9. Allows for validation of your system with the CRISPR/Cas9 system. A positive result in a miss-match detection assay will indicate validation of your system.

General description

Validated CRISPR site, which serves as an experimental control for the Wt Cas9. A two component positive control system consisting of a CMV-driven Cas9 plasmid and a U6-driven guide RNA plasmid targeting the human EMX1 gene.

Legal Information

CRISPR Use License AgreementLentiviral and WPRE License Agreements

Other Notes

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

Physical form

Sigma U6-gRNA plasmid expressing a guide sequence to human EMX1 supplied at a concentration of 20ng/ul in 50ul. Sigma Cas9 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-preppingplasmids 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.