pAAVS1D-CMV-RFP-EF1α-copGFPpuro PrecisionX™ HR Targeting Vector
Knock-in RFP into the AAVS1 site with our first-generation positive control AAVS1 HR Targeting Vector—pAAVS1D-CMV-RFP-EF1α-copGFPpuro
Take advantage of the power of the AA VS1 Safe Harbor Site
When you want a positive control to validate AAVS1 genomic locus targeting or if you’d like to make fluorescently labeled cells with RFP expressed from the AAVS1 site, use this ready-to-transfect first generation pAAVS1D-CMV-RFP-EF1α-copGFPpuro PrecisionX HR Targeting Vector. To insert RFP at the AAVS1 site, simply co-transfect with Cas9 and gRNA delivery constructs, such as our All-in-one Cas9 SmartNuclease & AAVS1 gRNA Plasmid.
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.
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.
Why use this vector?
The pAAVS1D-CMV-RFP-EF1α-copGFPpuro AAVS1 HR Targeting Vector is ready-to-transfect, with RFP driven by the strong CMV promoter. This vector also features dual GFP and Puromycin-resistance markers driven by a constitutive EF1α promoter, enabling easy monitoring of successful recombination by GFP fluorescence, as well as rapid selection for integration using the puromycin antibiotic marker.
How It Works
Genome engineering with CRISPR/Cas9
For general guidance on using CRISPR/Cas9 technology for genome engineering, take a look at our CRISPR/Cas9 tutorials as well as the following application notes:
Through careful selection of the target sequence and design of a donor plasmid for homologous
recombination, you can achieve efficient and highly targeted genomic modification with CRISPR/Cas9.
Cas9 protein—uses guide RNA (gRNA) to direct site-specific, double-strand DNA cleavage adjacent to a protospacer adapter motif (PAM) in the target DNA.
gRNA—RNA sequence that guides Cas9 to cleave a homologous region in the target genome. Efficient cleavage only where the gRNA homology is adjacent to a PAM.
PAM—protospacer adapter motif, NGG, is a target DNA sequence that spCas9 will cut upstream from if directed to by the gRNA.
The workflow at-a-glance
DESIGN: Select gRNA and HR donor plasmids. Choice of gRNA site and design of donor
plasmid determines whether the homologous recombination event results in a knock-out,
knock-in, edit, or tagging.
CONSTRUCT: Clone gRNA into all-in-one Cas9 vector. Clone 5’ and 3’ homology arms into HR
donor plasmid. If creating a knock-in, clone desired gene into HR donor.
CO-TRANSFECT or CO-INJECT: Introduce Cas9, gRNA, and HR Donors into the target cells
using co-transfection for plasmids, co-transduction for lentivirus, or co-injection for mRNAs.
SELECT/SCREEN: Select or screen for mutants and verify.
VALIDATE: Genotype or sequence putative mutants to verify single or biallelic conversion.