A kit that streamlines reporter cell line creation from the powerful AAVS1 site
Get the critical reagents for creating a reporter cell line that expresses GFP from your promoter-of-interest at the AAVS1 Safe Harbor Site. This Kit bundles together an HR Donor—the AAVS1-Targeting Reporter Knock-in HR Donor Vector 2.0 AAVS1-SA-puro-MCS-GFP—a Cas9/AAVS1 gRNA delivery construct—the All-in-one Cas9 SmartNuclease & AAVS1 gRNA Plasmid—and a 5’ and 3’ primer mix for a Junction PCR Assay to verify integration into the AAVS1 site.
Our second generation, Reporter Knock-in HR Donor Vector 2.0 AAVS1-SA-puro-MCS-GFP contains a multiple cloning site (MCS) upstream of a promoterless GFP, enabling creation of a reporter cell line that expresses GFP from the AAVS1 locus using the promoter of your choice. Like all of our AAVS1 HR Donor Vectors, it come with AAVS1 homology arms already cloned in, simplifying your workflow. Just ligate-in the promoter of your choice and co-transfect with the included All-in-one Cas9 SmartNuclease & AAVS1 gRNA Plasmid. You can verify correct insertion with the included 5’ and 3’ Primer Mix for Junction PCR Assay.
Why choose one of our second generation AAVS1-targeting HR Donors?
The clever design of our second generation AAVS1-Targeting HR Donor Vectors limits off-target integration for highly-specific targeting of the AAVS1 site. Taking advantage of the AAVS1’s location within an intron, the puromycin marker has only a splice acceptor site and no promoter. Expression of puromycin can only occur when the construct integrates within an intron, reducing the probability of recovering off-target integrants in the presence of puromycin selection.
Delivering consistent, robust transgene expression, the AAVS1 safe harbor site is a preferred target for gene knock-ins. Insertion at the site has been shown to be safe with no phenotypic effects reported, and the surrounding DNA appears to be kept in an open confirmation, enabling stable expression of a variety of transgenes.
SBI’s AAVS1 Safe Harbor Targeting products deliver:
- Easy, precise knock-in of any gene
- Consistent, robust transgene expression from the AAVS1 Safe Harbor Site
- Simplified construction of isogenic cell lines
- Minimal off-target integration
- Streamlined CRISPR/Cas9 library screening
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.