PiggyBac Gene Editing HR Targeting Vector (MCS1-5'PB TR-EF1α-GFP-T2A-Puro-T2A-hsvTK-pA-3' PB TR-MCS2)

Without a trace—make seamless gene edits with no residual footprint—includes dual GFP/puromycin selection and on-target enrichment with TK selection

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piggyBac-HR with GFP+Puro markers and TK selection (MCS1-5’PB TR-EF1α-GFP-T2A-Puro-T2A-hsvTK-pA-3′ PB TR-MCS2) for Gene Editing

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piggyBac-HR with GFP+Puro markers and TK selection (MCS1-5’PB TR-EF1α-GFP-T2A-Puro-T2A-hsvTK-pA-3′ PB TR-MCS2) for Gene Editing

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10 µg
PBHR100A-1
$ 1152
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Overview

Seamless gene editing

Use the PiggyBac Gene Editing HR Targeting Vector (MCS1-5’PB TR-EF1α-GFP-T2A-Puro-T2A-hsvTK-pA-3′ PB TR-MCS2) to get seamless gene editing—leave no trace of vector sequences behind.

This special HR Donor works with the Excision-only PiggyBac Transposase instead of the Cre-LoxP system. Simply proceed with your CRISPR/Cas9 gene editing as usual—clone your homology arms into MCS1 and MCS2, use dual GFP and puromycin selection to find integrants, and enrich for on-target events using negative thymidine kinase (TK) selection (Figure 1).

 

PiggyBac Gene Editing HR Targeting Vector (MCS1-5'PB TR-EF1α-GFP-T2A-Puro-T2A-hsvTK-pA-3' PB TR-MCS2)

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

Catalog # HR Donor Vector Features* Application
Gene Knock-out Gene Knock-in Gene Edits Gene Tagging
HR100PA-1 MCS1-LoxP-MCS2-MCS3-pA-LoxP-MCS4 Basic HR Donor
HR110PA-1 MCS1-EF1α-RFP-T2A-Puro-pA-MCS2 Removable RFP marker and puromycin selection
HR120PA-1 GFP-pA-LoxP-EF1α-RFP-T2A-Puro-pA-LoxP-MCSPuro-pA-LoxP-MCS Tag with GFP fusion
Removable RFP marker and puromycin selection
HR130PA-1 T2A-GFP-pA-loxP-EF1α-RFP-T2A-Puro-pA-LoxP-MCSA-loxP-EF1α-RFP-T2A-Puro-pA-LoxP-MCS Co-express GFP with “tagged” gene via T2A
Removable RFP marker and puromycin selection
HR150PA-1 GFP-T2A-Luc-pA-loxP-EF1α-RFP-T2A-Puro-pA-LoxP-MCS Tag with GFP fusion and co-express luciferase via T2A
Removable RFP marker and puromycin selection
HR180PA-1 IRES-GFP-pA-loxP-MCS1-EF1α-RFP-T2A-Puro-pA-LoxP-MCS2 Co-express GFP with “tagged” gene via IRES
Removable RFP marker and puromycin selection
HR210PA-1 MCS1-LoxP-EF1α-GFP-T2A-Puro-P2A-hsvTK-pA-LoxP-MCS2 Removable GFP marker, puromycin selection, and TK selection
HR220PA-1 GFP-pA-LoxP-EF1α-RFP-T2A-Hygro-pA-LoxP-MCS Tag with GFP fusion
Removable RFP ,arker and hygromycin Selection
HR410PA-1 MCS1-EF1α-GFP-T2A-Puro-pA-MCS2 Removable GFP marker and puromycin selection
HR510PA-1 MCS1-EF1α-RFP-T2A-Hygro-pA-MCS2 Removable RFP marker and hygromycin selection
HR700PA-1 MCS1-EF1α-GFP-T2A-Puro-pA-MCS2-PGK-hsvTK Enrich for on-target integration with negative TK selection**
Removable GFP marker and puromycin selection
HR710PA-1 MCS1-EF1α-RFP-T2A-Hygro-pA-MCS2-PGK-hsvTK Enrich for on-target integration with negative TK selection**
Removable RFP marker and hygromycin selection
HR720PA-1 MCS1-EF1α-Blasticidin-pA-MCS2-PGK-hsvTK Enrich for on-target integration with negative TK selection**
Removable blasticidin selection
GE602A-1 pAAVS1D-PGK-MCS-EF1α-copGFPpuro First generation AAVS1-targeting HR Donor
GE603A-1 pAAVS1D-CMV-RFP-EF1α-copGFPpuro First generation AAVS1-targeting HR Donor (positive control for GE602A-1)
GE620A-1 AAVS1-SA-puro-MCS Second generation AAVS1-targeting HR Donor
Promoterless to knock-in any gene or promoter-gene combination
GE622A-1 AAVS1-SA-puro-EF1α-MCS Second generation AAVS1-targeting HR Donor
Constitutive expression of your gene-of-interest
GE624A-1 AAVS1-SA-puro-MCS-GFP Second generation AAVS1-targeting HR Donor
Create reporter cell lines
CAS620A-1 AAVS1-SA-puro-EF1α-hspCas9 Knock-in Cas9 to the AAVS1 site
PBHR100A-1 MCS1-5'PB TR-EF1α-GFP-T2A-Puro-T2A-hsvTK-pA-3' PB TR-MCS2 Use with the PiggyBac Transposon System
Enables seamless gene editing with no residual footprint (i.e. completely remove vector sequences)
*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.

How It Works

Getting seamless gene editing with the Excision-only PiggyBac Transposase

Seamless gene editing with the Excision-only PiggyBac Transposase

Figure 1. Seamless gene editing with the Excision-only PiggyBac Transposase. Step 1: Cas9 creates a double-stranded break (DSB) in the genomic DNA at a site that is complimentary to the gRNA. For gene editing, this DSB should be within an intron. 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 vector DNA) homologous recombination (HR) is favored over non-homologous end joining (NHEJ). If one of the homology arms of the HR donor contains the gene edit, it will be incorporated into the gene through the HR repair process. Step 3: Like the Cre-LoxP system, the PiggyBac Transposon system relies on an enzyme—the transposase—to mediate a site-specific recombination event between two sites, the 5’ PiggyBac Terminal Repeat (TR) and the 3’ PiggyBac TR. However, unlike the Cre-LoxP system, the Excision-only PiggyBac Transposase completely removes the 5’ and 3’ PiggyBac TRs as well as any sequences in between the two TRs. The design of the PiggyBac Gene Editing HR Targeting Vector (MCS1-5’PB TR-EF1α-GFP-T2A-Puro-T2A-hsvTK-pA-3′ PB TR-MCS2) places two homology arms just outside of the PiggyBac Terminal Repeats (TRs). Thus, after Excision-only PiggyBac Transposase activity, all vector sequences are removed.

Genome engineering with CRISPR/Cas9

For general guidance on using CRISPR/Cas9 technology for genome engineering, including the design of HR Targeting Vectors, 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) »


Citations

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