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Human AAVS1 safe harbor gene knock-in kits and clones

 

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Introduction

AAVS1 (also known as the PPP1R12C locus) on human chromosome 19 is a well-validated “safe harbor” for hosting DNA transgenes with expected function. It has an open chromatin structure and is transcription-competent. Most importantly, there are no known adverse effects on cells resulting from the inserted DNA fragment of interest.

AAVS1-specific TALENs or CRISPR-Cas9 sgRNAs generate DNA double-strand breaks (DSBa) at the AAVS1 locus, stimulating DNA repair. In the presence of AAVS1 ORF knockin clones, homologous recombination (HR) occurs and integrates the ORF knockin into the safe harbor locus.

 
 
 
 

Human AAVS1 Safe Harbor knockin kits

Buy Catalog # Product Description Price
SH-AVS-K100 Genome-TALER™ human AAVS1 safe harbor gene knock-in kit Includes: AAVS1 TALEN pair (TN-AAVS1) AAVS1 donor cloning vector (DC-DON-SH01) AAVS1 positive control donor (DC-RFP-SH01) knock-in verification primer pairs (HQPAVSHR) $1895
SH-AVS-K000 Genome-TALER™ human AAVS1 safe harbor gene knock-in kit (without donor) Includes: AAVS1 TALEN pair (TN-AAVS1) AAVS1 positive control (DC-RFP-SH01) knock-in verification primer pairs (HQPAVSHR) $1695
SH-AVS-K200 Genome-CRISP™ human AAVS1 safe harbor gene knock-in kit Includes: AAVS1 All-in-one sgRNA/Cas9 expression clone (HCP-AAVS1-CG02) AAVS1 donor cloning vector (DC-DON-SH01) AAVS1 positive control donor (DC-RFP-SH01) knock-in verification primer pairs (HQPAVSHR) $1895
SH-AVS-K002 Genome-CRISP™ human AAVS1 safe harbor gene knock-in kit (without donor) Includes: AAVS1 All-in-one sgRNA/Cas9 expression clone (HCP-AAVS1-CG02) AAVS1 positive control donor (DC-RFP-SH01) knock-in verification primer pairs (HQPAVSHR) $1695

 

Human AAVS1 Safe Harbor knockin ORF clones

Catalog # Product Description Price
Varies AAVS1 knockin ORF donor clone AAVS1 knockin ORF donor clone containing CMV-driven ORF of customer's gene of interest  

 

Human AAVS1 Safe Harbor knockin CRISPR-Cas9 clones

Important Note: The vector-specifc knock-in verification primers provided with the Human AAVS1 Safe Harbor knockin kits (catalog numbers SH-AVS-K100, SH-AVS-K000, SH-AVS-K200, and SH-AVS-K002) are not compatible with the Human AAVS1 Safe Harbor knockin CRISPR-Cas9 clones in the table below. If you purchase these clones with our Human Safe Harbor knockin kits, you will be provided with clone-specific knockin verification primers.
 
Buy Catalog # Product Application Selection marker Promoter Cas9 integration site Delivery format Price
DC-C9NU-03 Cas9 AAVS1 knockin donor clone To knock in Cas9 nuclease to human AAVS1 Safe Harbor site using CRISPR or TALEN Puro CBh AAVS1 Bacterial stock or transfection-ready DNA $495
DC-C9NU-04 Cas9 AAVS1 knockin donor clone Hygro CBh AAVS1 $495
DC-C9NU-05 Cas9 AAVS1 knockin donor clone Neo CBh AAVS1 $495
 
   
TALEN kits The kits are designed to specifically transfer your gene of interest, selection marker or other genetic element into the AAVS1 safe harbor site in human chromosome 19 via TALEN-mediated homologous recombination. The Genome-TALER™ human AAVS1 safe harbor gene knock-in kit includes one pair of AAVS1 TALENs, one donor cloning vector or one donor clone*, one positive control clone and two pairs of knock-in PCR verification primers.
(A)  
AAVS1_Left_TALEN-m AAVS1_Right_TALEN-m
     
AAVS1_Donor_clone-m AAVS1_Donor_control-m  
(B)
  Figure 1. Genome-TALER™ human AAVS1 safe harbor gene knock-in kit. (A) AAVS1 TALENs and donor DNA plasmids (B) AAVS1 knock-in verification PCR primers  
Cat. No. Components Description Qty
 TN-AAVS1-L  AAVS1 left TALEN Specifically cleave AAVS1 with target site TN-AAVS1-R to create DSB 1
 TN-AAVS1-R  AAVS1 right TALEN Specifically cleave AAVS1 with target site TN-AAVS1-L â€‹to create DSB 1
 DC-DON-SH01*  AAVS1 donor cloning vector

For cloning GOI or containing GOI

–Two AAVS1 flanking arms for HR

-CMV promoter

-GFP/Puro marker for selection

1
 DC-RFP-SH01 AAVS1 positive control donor clone RFP instead of GOI for positive control purpose 1
 HQPAVSHR-5 5' knock-in verification PCR primer pair PCR primer pairs for 5’ HR junctions to verify successful integration 1
 HQPAVSHR-3 3' knock-in verification PCR primer pair PCR primer pairs for 3’ HR junctions to verify successful integration 1
* DC-DON-SH01 only comes with SH-AVS-K100 kit. Genome-TALER™ AAVS1 ORF knock-in clones can be  and ordered separately. Other AAVS1 donor clones can also be  by us.   
(A) (B)
The_comparison_chart1-s
(C)
(D)
  Figure 2. Human genome safe harbor AAVS1 gene targeting (A) AAVS1 RFP control plasmid DC-RFP-SH01 (800 ng) was co-transfected with AAVS1 TALEN pair(600 ng for each) or control TALEN pair into HEK293T cells in a 6-well plate. (B) 48 hr post-transfection, the cells were split 1:10 into a new 6-well plate and selected against 1.0 µg/ml of puromycin. The images were taken after two weeks of selection. Few colonies left in the wells transfected with AAVS1 RFP control + control TALEN.  (C) (D) PCR primers designed to amplify the HR junction were used to verify the specific and successful integration.
  Acknowledgement: Design of the AAVS1 left TALEN, AAVS1 right TALEN, and AAVS1 donor control vectors was performed by Dr. Jizhong Zou of the NIH Center for Regenerative Medicine, a Common Fund initiative of the U.S. National Institutes of Health.
CRISPR kits The kits are designed to specifically transfer your gene of interest, selection marker or other genetic element into the AAVS1 safe harbor site in human chromosome 19 via CRISPR-Cas9-mediated homologous recombination. The Genome-CRISP™ human AAVS1 safe harbor gene knock-in kit includes one AAVS1 sgRNA/Cas9 expression clone, one donor cloning vector or one donor clone*, one positive control clone and one knock-in PCR verification kit.  
(A)    

AAVS1_Donor_clone-m

     
AAVS1_Donor_clone-m AAVS1_Donor_control-m  
(B)
Figure 3. Genome-CRISP™ human AAVS1 safe harbor gene knock-in kit. (A) AAVS1 sgRNA/Cas9 expression clone and donor DNA plasmids (B) AAVS1 knock-in verification PCR primers  
Cat. No. Components Description Qty
 HCP-AAVS1-CG02  AAVS1 sgRNA/Cas9 expression clone Specifically cleave AAVS1 target site to create DSB 1
 DC-DON-SH01*  AAVS1 donor cloning vector

For cloning GOI or containing GOI

–Two AAVS1 flanking arms for HR

-CMV promoter

-GFP/Puro marker for selection

1
 DC-RFP-SH01 AAVS1 positive control donor clone RFP instead of GOI for positive control purpose 1
 HQPAVSHR-5 5' knock-in verification PCR primer pair PCR primer pairs for 5’ HR junctions to verify successful integration 1
 HQPAVSHR-3 3' knock-in verification PCR primer pair PCR primer pairs for 3’ HR junctions to verify successful integration 1

* DC-DON-SH01 only comes with SH-AVS-K200 kit. Genome-TALER™ AAVS1 ORF knock-in clones can be  and ordered separately. Other AAVS1 donor clones can also be  by us.

Knock-in clones Human AAVS1 safe harbor knock-in ORF clones More than 20,000 sequence-validated human AAVS1 safe harbor knock-in ORF clones are available for you to . These clones are constructed for specifically integrating the ORF of your interest from a donor plasmid into the AAVS1 safe harbor site in human chromosome 19 by using Genome-TALER™ or Genome-CRISP™ human AAVS1 safe harbor gene knock-in kits.

AAVS1_Donor_clone-m

Figure 4. Map of human AAVS1 safe harbor knock-in ORF clones

 

Human AAVS1 Safe Harbor knockin CRISPR-Cas9 clones

Buy Catalog # Product Application Selection marker Promoter Cas9 integration site Delivery format Price
DC-C9NU-03 Cas9 AAVS1 knockin donor clone To knock in Cas9 nuclease to human AAVS1 Safe Harbor site using CRISPR or TALEN Puro CBh AAVS1 Bacterial stock or transfection-ready DNA $495
DC-C9NU-04 Cas9 AAVS1 knockin donor clone Hygro CBh AAVS1 $495
DC-C9NU-05 Cas9 AAVS1 knockin donor clone Neo CBh AAVS1 $495
  Inducible Cas9 AAVS1 knockin donor clone   Puro TRE3G AAVS1   $495
  Inducible Cas9 AAVS1 knockin donor clone Blast TRE3G AAVS1 $495
  Inducible Cas9 AAVS1 knockin donor clone Hygro TRE3G AAVS1 $495
 

Knock-in clone custom services also available

GeneCopoeia also offers AAVS1 donor clone custom services to construct knock-in donor clones containing mutated genes or other genetic elements for targeted AAVS1 integration. To get a quote, please .

Questions? Please contact us at inquiry@genecopoeia.com or 1-866-360-9531.

Figure 5: (A) OCT4 (Cat#: DC-Z0092-SH01) or SOX2(Cat#: DC-T2547-SH01) donor plasmids were co-transfected  with AAVS1 TALEN pair into HEK293T cells in a 6-well plate.  Cells were sub-cultrured 48 hr post-transefection and selected against puromycin (1µg/ml) for  2 weeks.  The expression of CopGFP was checked under a microscope (Nikon Eclipse Ti ) 48h post-transfection  or after 2 weeks of drug selection.  Few colonies left in cells transfected with donor plasmid alone after puromycin selection (data not shown). (B) Western blot analysis of proteins from HEK293T cells stably integrated with SOX2(Cat#: DC-T2547-SH01) or OCT4 (Cat#: DC-Z0092-SH01) in AAVS1 site, with cells alone as negative control where endogenous SOX2  or OCT4 protein levels were too low to be detected in the same blot.  

 

Services

AAVS1 donor clone custom services

GeneCopoeia also offers AAVS1 donor clone custom services to construct knock-in donor clones containing mutated genes or other genetic elements for targeted AAVS1 integration. To get a quote, please .

Questions? Please contact us at inquiry@genecopoeia.com or 1-866-360-9531.

 

AAVS1 stable cell line custom services

Services Description Application
Stable cell line services Monoclonal colony Monoclonal stable cell line with TALEN- or CRISPR-Cas9-mediated safe harbor transgene. human AAVS1
Cell bank Create cell bank of monoclonal stable cell line with TALEN or CRISPR-Cas9-mediated safe harbor transgene. human AAVS1

  Questions? Please contact us at inquiry@genecopoeia.com or 1-866-360-9531.

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