Model EF1a-hspCas9-nickase-H1-gRNA - All-in-one Cas9 SmartNickase Plasmid (circular)

Stay on-target with your genome editing projects

For pre-clinical and other applications where you need to minimize off-target Cas9 activity and are using transfectable cells, All-in-one Cas9 SmartNickase plasmids are an excellent choice. Unlike our newer Cas9 constructs, this first generation Cas9 SmartNickase—EF1α-hspCas9-nickase-H1-gRNA All-in-one Cas9 SmartNickase Plasmid—does not contain a selectable marker for identifying transfected cells and is now available as an intact, circular plasmid.

Unlike the wildtype Cas9 protein which introduces double-strand breaks (DSBs), the Cas9 SmartNickase introduces paired nicks at the gRNA-directed site. Creating nicks favors the higher-fidelity homologous recombination process over non-homologous end joining (NHEJ), with paired nicking shown to reduce off-target activity by 50- to 1,500-fold in cell lines, and to facilitate gene knockout in mice without losing on-target cleavage efficiency1.

• Conveniently deliver Cas9 Nickase and gRNA with a single vector
• Reduce off-target activity
• Drive Cas9 Nickase expression with the EF1α promoter, which provides medium expression levels in most cell types, including primary cells and stem cells
• Express gRNA from the H1 promoter for maximum specificity and choice of targets
• Ensure efficient import of Cas9 Nickase to the nucleus with N-term and C-term nuclear localization signals (NLSs)
• Boost Cas9 Nickase gene expression and stabilize the transcript via the WPRE regulatory element after the C-term NLS
• Easily detect and/or purify the Cas9 Nickase protein with the N-term myc-tag

As with all of our Cas9 Nickase delivery options, the EF1α-hspCas9-nickase-H1-gRNA Plasmid is functionally validated and comes backed by our expert technical support team—if you’ve got a genome engineering question just ask by emailing tech@systembio.com.

Why an HR targeting vector is a recommended

Even though gene knock-outs can result from DSBs caused by Cas9 alone, SBI recommends the use of HR targeting vectors (also called HR donor vectors) for more efficient and precise mutation. HR donors can supply elements for positive or negative selection ensuring easier identification of successful mutation events. In addition, HR donors can include up to 6-8 kb of open reading frame for gene knock-ins or tagging, and, when small mutations are included in either 5’ or 3’ homology arms, can make specific, targeted gene edits.