Gene Knock-Out HR Targeting Vector w/Single Selection Marker (Blasticidin) and Negative Selection (TK) Against Random Integration

Select against off-target events with OnTarget™ HR Targeting Vectors Use the PrecisionX™ OnTarget™ Gene Knock-out HR Targeting Vector (MCS1-EF1α-Blasticidin-pA-MCS2-PGK-hsvTK) to knock-out any gene or edit the genome. Clone your homology arms into MCS1 and MCS2, and use blasticidin selection to find integrants. Even better, with this series of OnTarget™ HR Donors, you can enrich for on-target events using negative thymidine kinase (TK) selection (Figure 1). After you’ve identified clones with your gene-of-interest knocked-out or edited, you can remove the selection cassette using the Cre-LoxP system (learn more about Cre-LoxP excision here).

 

 

Figure 1. Using negative TK selection to enrich for on-target events . With on target HR Donor-mediated gene editing (left column), the homology arms of the HR Donor drive homologous recombination, resulting in insertion of only the region of the HR Donor that lies between the two homology arms—if the PGK-hsvTK cassette is outside of the homology arms, it will not be inserted into the genome and cells will not be sensitive to ganciclovir or fialuridine.

However, with off target insertion of the HR Donor (right column), integration is not mediated by homology, resulting in insertion of the entire HR Donor Vector. If the HR Donor includes the PGK-hsvTK cassette, even if the cassette is outside of the homology arms, the cassette will get inserted into the genome, rendering the cells sensitive to ganciclovir or fialuridine and enabling negative selection against off-target events.

Why use an HR targeting vector?

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.

Choose the right HR Targeting Vector for your project

*All HR Target Vectors except PBHR100A-1 contain LoxP sites. Any sequences that are integrated between the two LoxP sites can be removed through transient expression of Cre Recombinase.
**The clever design of these HR Donors enables enrichment for on-target integration events. A PGK-hsvTK cassette is included outside of the homology arms. Because of this configuration, on-target integration that results from homologous recombination will not include the PGK-hsvTK cassette—only randomly-integrated off-target events will lead to integration of PGK-hsvTK and resulting TK activity. Therefore, TK selection will negatively select against off-target integrants. Click on any one of these vectors to see a diagram of how the negative selection works.]