CRISPR Cas9-D10A Nickase Plasmid

The CRISPR Cas9-D10A Nickase 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-D10A expression plasmids can be linearized using XbaI for T7-based mRNA production.

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

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.

Cas9-D10A single vector can be expanded for use with any U6-guideRNA plasmids. Amounts of Cas9 and guideRNA can be altered to optimize the ratio of Cas9 to guideRNA. Recent evidence indicates off-targeting by CRISPR endonucleases is a significant concern. To address this problem, Sigma has developed paired nickase technology to expand CRISPR DNA recognition tracts to lengths similar to those of ZFNs and TALENs.  Our experience suggests that paired nickases based on the Cas9-D10A mutant are most reliable. Our development work also suggests that the critical factor in producing active paired nickases is the positioning of gRNAs in a 5′-to-5′ orientation. For plasmid-based delivery of paired CRISPR nickases, we recommend the use of a three plasmid system comprised of the Cas9-D10A expression vector and two U6-driven gRNA vectors. To simplify the use of paired nickases, Sigma has on-line tools which allow access to a library of pre-designed paired nickases. Please check Sigma′s on-line CRISPR product offerings for the latest design sets.

Sigma Cas9-D10A Nickase plasmid DNA is supplied at concentrations of 20ng/ul in 50ul.

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.

1 vial containing 1ug of Cas9-D10A Nickase plasmid.

Please note, product does not contain guideRNA sequence. This must be purchased separately through the Custom CRISPR product tab.

Must be used in conjunction with two U6-gRNA plasmids 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-D10A plasmids combined with >= 1.0 ug/uL and <= 5 uL of U6-gRNA plasmids. (All dosages above assume 0.5 to 1 million cells nucleofected)