Name: AAVS1 Safe Harbor Targeting Vector 2.0 - Reporter Knock-in Donor (AAVS1-SA-puro-MCS-GFP), Complete Kit with CAS601A-1 (Cas9 SmartNuclease AAVS1-gRNA Targeting Vector) and GE640PR-1 (Junction PCR Primer Mix to confirm AAVS1 integration site)
Brand: System Biosciences
SKU: GE620A-KIT
Price: 2402 EUR
Availability: InStock

AAVS1 Safe Harbor Targeting Vector 2.0 - Reporter Knock-in Donor (AAVS1-SA-puro-MCS-GFP), Complete Kit with CAS601A-1 (Cas9 SmartNuclease AAVS1-gRNA Targeting Vector) and GE640PR-1 (Junction PCR Primer Mix to confirm AAVS1 integration site)

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SKU:

GE620A-KIT

Availability:

Usually Shipped in 5 Working Days

Size:

1 kit

Shipping Temperature:

RT/Blue Ice/ Dry Ice

Description

Description

Easily create reporter cell lines that express GFP from the AAVS1 Site—includes HR Donor, All-in-one Cas9 & AAVS1 gRNA Vector, and verification primer mix

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

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.

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 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.

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