AAVS1 Safe Harbor Site Targeting 2.0 Complete Kit with All-purpose HR Donor Vector 2.0 (AAVS1-SA-puro-MCS-GFP)
- 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
Products
| Catalog Number | Description | Size | Price | Quantity | Add to Cart | |||
|---|---|---|---|---|---|---|---|---|
| GE624A-KIT | AAVS1 Safe Harbor Targeting 2.0 Reporter Knock-in Kit with GE624A-1 (AAVS1 Safe Harbor Targeting Reporter Knock-in HR Donor 2.0), CAS601A-1 (All-in-one Cas9 SmartNuclease AAVS1 Targeting Plasmid) and GE640PR-1 (Junction PCR Primer Mix to confirm AAVS1 integration site) | 1 Kit | $2111 |
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Overview
Overview
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.
Why AAVS1?
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.
References
How It Works
How It Works
Gene knock-in at AAVS1
Figure 1. Knocking-in a gene at the AAVS1 site using an HR Targeting Vector. Step 1: Cas9 creates a double-stranded break(DSB) at the AAVS1 site. Cas9 activity is directed to the AAVS1 site by an AAVS1-specific gRNA. Step 2: The DNA repair machinery is recruited to the DSB. In the presence of an HR Donor with homology to the region adjacent to the DSB (blue areas of the genomic and plasmid DNA) homologous recombination (HR) is favored over non-homologous end joining (NHEJ). Result: The HR event leads to insertion of the region of the HR Donor Vector between the two homology arms—your gene-of-interest is integrated into the AAVS1 site.
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:
CRISPR/Cas9 Gene Knock-Out Application Note (PDF) »
CRISPR/Cas9 Gene Editing Application Note (PDF) »
CRISPR/Cas9 Gene Tagging Application Note (PDF) »
CRISPR/Cas9 Basics
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.
The system
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.
Supporting Data
FAQs
Resources
Citations
Related Products
Products
| Catalog Number | Description | Size | Price | Quantity | Add to Cart | |||
|---|---|---|---|---|---|---|---|---|
| GE624A-KIT | AAVS1 Safe Harbor Targeting 2.0 Reporter Knock-in Kit with GE624A-1 (AAVS1 Safe Harbor Targeting Reporter Knock-in HR Donor 2.0), CAS601A-1 (All-in-one Cas9 SmartNuclease AAVS1 Targeting Plasmid) and GE640PR-1 (Junction PCR Primer Mix to confirm AAVS1 integration site) | 1 Kit | $2111 |
|
||||
Overview
Overview
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.
Why AAVS1?
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.
References
How It Works
How It Works
Gene knock-in at AAVS1
Figure 1. Knocking-in a gene at the AAVS1 site using an HR Targeting Vector. Step 1: Cas9 creates a double-stranded break(DSB) at the AAVS1 site. Cas9 activity is directed to the AAVS1 site by an AAVS1-specific gRNA. Step 2: The DNA repair machinery is recruited to the DSB. In the presence of an HR Donor with homology to the region adjacent to the DSB (blue areas of the genomic and plasmid DNA) homologous recombination (HR) is favored over non-homologous end joining (NHEJ). Result: The HR event leads to insertion of the region of the HR Donor Vector between the two homology arms—your gene-of-interest is integrated into the AAVS1 site.
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:
CRISPR/Cas9 Gene Knock-Out Application Note (PDF) »
CRISPR/Cas9 Gene Editing Application Note (PDF) »
CRISPR/Cas9 Gene Tagging Application Note (PDF) »
CRISPR/Cas9 Basics
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.
The system
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.