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Safe Harbor Gene Knock-in Kits and Clones

Introduction

GeneCopoeia's Safe Harbor system is the ideal solution for expressing transgenes in cells and animals. Transgene insertion at the AAVS1 and ROSA26 sites in humans and mice, respectively, permits consistent and stable expression with no adverse effects on fitness. 

GeneCopoeia offers safe harbor knockin kits and clones as well as safe harbor premade cell lines. 

Advantages

  • Safe integration. Transgene integration at well-characterized safe harbor sites ensures consistent, stable expression with no adverse effects on cell viability or fitness.
  • Comprehensive system. CRISPR or TALEN-based kits come with a choice of donor cloning vectors, premade knockin clones, or made-to-order knockin clones, as well as verification PCR primers.
  • Compatible knock-in ORFs. Choose from more than 20,000 human and more than 15,000 mouse sequence-verified ORFs in custom-built knock-in donor clones.

 

Order Safe Harbor Knockin kits

Safe harbor kits contain the following major components: (1) AAVS1 or ROSA26 site-specific CRISPR or TALEN pair, (2) knockin junction verification primers and (3) a donor cloning vector or a donor clone. We currently offer three donor options: 

  • Do-it-yourself cloning vectors. For creating safe harbor knockin donor clones carrying any transgene you want.
  • Pre-made Cas9 donor clones. Express CRISPR-Cas9 nuclease from the safe harbor site.
  • ORF knockin clones. Made-to-order from GeneCopoeia's vast, searchable collections of more than 20,000 human and more than 15,000 mouse safe harbor knockin ORFs.

 

To display your kits of interest, select species, genome editing technology, and donor options from the table below. 

Species

CRISPR or TALEN

Donor


 

 

Order pre-made Cas9 stable cell lines

GeneCopoeia also offers stable cell lines constitutively expressing the CRISPR Cas9 nuclease, enabling you to carry out CRISPR genome editing applications with high efficiency.

The Genome-CRISP™ Cas9 stable cell lines are available pre-made in many human cell lines, and in mouse cell line Neuro2a. The CRISPR Cas9 nuclease is stably integrated into either the human AAVS1 or mouse ROSA26 “Safe Harbor” sites (Figure 1), or randomly via lentiviral-mediated transduction.

Choose your human or mouse Cas9-expressing stable cell line from the list below. For more information, please vist our Cas9 stable cell lines page.

Buy Catalog # Cell line Description Selection marker Cas9 integration site Price, Academic Price, Industry
Human Cas9-expressing stable cell lines
SL501 H1299 Human cell line H1299 stably expressing CRISPR Cas9, single clone Puro AAVS1 $1395 $2695
SL502 HEK293T Human cell line HEK293T stably expressing CRISPR Cas9, single clone Puro AAVS1 $945 $1995
SL503 HeLa Human cell line HeLa stably expressing CRISPR Cas9, single clone Hygro AAVS1 $1395 $2695
SL504 A549 Human cell line A549 stably expressing CRISPR Cas9, single clone Hygro AAVS1 $1395 $2695
SL514 MCF-7 Human cell line MCF-7 stably expressing CRISPR Cas9, single clone Hygro AAVS1 $1395 $2695
SL515 MDA-MB-231 Human cell line MDA-MB-231 stably expressing CRISPR Cas9, single clone Hygro Random $1395 $2695
SL516 MDA-MB-468 Human cell line MDA-MB-468 stably expressing CRISPR Cas9, single clone Hygro Random $1395 $2695
SL517 T47D Human cell line T47D stably expressing CRISPR Cas9, single clone Hygro Random $1395 $2695
SL518 HepG2 Human cell line HepG2 stably expressing CRISPR Cas9, single clone Puro AAVS1 $1395 $2695
SL520 AGS Human cell line AGS stably expressing CRISPR Cas9, single clone Hygro Random $1395 $2695
SL521 BXPC-3 Human cell line BXPC-3 stably expressing CRISPR Cas9, single clone Hygro Random $1395 $2695
SL522 SNU-475 Human cell line SNU-475 stably expressing CRISPR Cas9, single clone Hygro Random $1395 $2695
SL523 HT-29 Human cell line HT-29 stably expressing CRISPR Cas9, single clone Hygro Random $1395 $2695
SL525 LoVo Human cell line LoVo stably expressing CRISPR Cas9, single clone Hygro Random $1395 $2695
SL527 KATO111 Human cell line KATO111 stably expressing CRISPR Cas9, single clone Hygro Random $1395 $2695
SL535 SK-BR-3 Human cell line SK-BR-3 stably expressing CRISPR Cas9, single clone Hygro Random $1395 $2695
SL544 HCC70 Human cell line HCC70 stably expressing CRISPR Cas9, single clone Hygro Random $1395 $2695
Mouse Cas9-expressing stable cell lines
SL509 Neuro2a Mouse cell line Neuro2a stably expressing CRISPR Cas9, single clone Hygro ROSA26 $1395 $2695
SL510 Neuro2a Mouse cell line Neuro2a stably expressing CRISPR Cas9, single clone Puro ROSA26 $1395 $2695
SL511 Neuro2a Mouse cell line Neuro2a stably expressing CRISPR Cas9, single clone Neo ROSA26 $1395 $2695

  MSDS

 

Note: GeneCopoeia also offers custom-built Cas9-expressing stable cell line services. To inquire, please contact inquiry@genecopoeia.com.

 

 

 

Order Individual Safe Harbor Components

Buy Catalog # Vector Description Selection marker Reporter gene Target site Price
SH100 pCRISPR-CG02 All-in-one (Cas9 + sgRNA) clone targeting human AAVS1 - - AAVS1 $895
SH125 pTALEN-01 TALEN pair targeting human AAVS1 - - AAVS1 $895
SH150 pCRISPR-CG01 All-in-one (Cas9 + sgRNA) clone targeting mouse ROSA26 Neo mCherry ROSA26 $895
SH175 pTALEN-01 TALEN pair targeting mouse ROSA26 - - ROSA26 $895

 

 

Buy Catalog# Name Description Type Selection marker Reporter gene Tag Price
SH200 DC-DON-SH01 DC-DON-SH01 expression vector for restriction enzyme cloning (AAVS1 knockin) Human Safe Harbor Puromycin copGFP - $425
EZ012 pEZ-SH01 pEZ-SH01 expression vector for LxR recombination cloning (AAVS1 knockin) Human Safe Harbor Puromycin copGFP - $425
SH250 DC-DON-SH02 DC-DON-SH02 expression vector for restriction enzyme cloning (ROSA26 knockin) Mouse Safe Harbor Puromycin copGFP - $425
EZ013 pEZ-SH02 pEZ-SH02 expression vector for LxR recombination cloning (ROSA26 knockin) Mouse Safe Harbor Puromycin copGFP - $425

 

 

Pre-made Safe Harbor knockin donor clones are designed for CRISPR- or TALEN-mediated knockin of the CRISPR Cas9 nuclease gene to the human AAVS1 or mouse ROSA26 sites. These clones are extremely valuble for several applications, notably lentiviral-mediated sgRNA delivery, sgRNA functional validation, and sgRNA library screening, and can be expressed either constitutively or from the Tet-inducible promoter. Each clone is provided with verification PCR primer pairs.

Buy Catalog # Product Description Selection marker Cas9 integration site Price
SH300 DC-C9NU-03 Cas9 AAVS1 knockin donor clone-Puro Puro AAVS1 $495
SH301 DC-C9NU-04 Cas9 AAVS1 knockin donor clone-Hygro Hygro AAVS1 $495
SH302 DC-C9NU-05 Cas9 AAVS1 knockin donor clone-Neo Neo AAVS1 $495
SH350 DC-C9NU-06 Cas9 ROSA26 knockin donor clone-Puro Puro ROSA26 $495
SH351 DC-C9NU-07 Cas9 ROSA26 knockin donor clone-Hygro Hygro ROSA26 $495
SH352 DC-C9NU-08 Cas9 ROSA26 knockin donor clone-Neo Neo ROSA26 $495

 

 

These pre-made Safe Harbor knockin donor clones express red fluorescent protein (RFP) from the human AAVS1 or mouse ROSA26 sites. Each clone is provided with verification PCR primer pairs.

Buy Catalog # Product Description Selection marker Cas9 integration site Price
SH308 DC-RFP-SH01 RFP AAVS1 knockin donor clone Puro AAVS1 $495
SH358 DC-RFP-SH02 RFP ROSA26 knockin donor clone Puro ROSA26 $495

 

 

Catalog # Product Description Price
Varies AAVS1/ROSA26 knockin ORF donor clone AAVS1/ROSA26 knockin ORF donor clone containing CMV-driven ORF of customer's gene of interest
GeneCopoeia also offers AAVS1 and ROSA26 donor clone custom services to construct knockin donor clones containing genetic elements for targeted Safe Harbor integration. To get a quote, please click here.
 

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

 

How it works

Background on Genome Editing and Safe Harbor

Genome modification by insertion of genes of interest and other genetic elements is of great value for cell engineering. Genetically modified cells are valuable for therapeutic research, gene function studies, and lineage tracking and analysis. Each of these applications depend on the reliable and predictable function of the transgene without causing adverse efffects on cells. However, random integration of the transgene, by traditional lentival methods, can occasionally disrupt genes inportant for cell function. In addition, random integration causes genes to be inserted as direct repeats, which can cause unwanted amplification of gene expression, and also lead to silencing of the gene over time.

Alternatively, transgenes can be inserted at specific sites in the genome.

The AAVS1 (also known as PPP1R12C locus) site on human chromosome 19, and ROSA26 site on mouse chromosome 6, are well-validated “safe harbor” loci for hosting DNA fragments. These sites have open chromatin structure and are transcription-competent. Most importantly, there are no known adverse effects on cells resulting from the inserted DNA fragment of interest.

The CRISPR and TALEN genome editing systems can be used to generate DNA double-strand breaks (DSB) at the AAVS1 and ROSA26 sites. In the presence of a homologous knockin donor, homologous recombination (HR) occurs, leading to integration of DNA fragments in the donor into the safe harbor sites (Figure 1).

 

 

Figure 1. CRISPR/TALEN-mediated transgene integration at safe harbor sites

GeneCopoeia's human AAVS1 and mouse ROSA26 Safe Harbor knockin kits are designed to specifically transfer your gene of interest, selection marker or other genetic element into these safe harbor sites via CRISPR- or TALEN-mediated homologous recombination.

CRISPR-based Human AAVS1 Safe Harbor system

     

AAVS1_Donor_clone-m

     
   
 

Figure 2. Genome-CRISP™ CRISPR-based human AAVS1 safe harbor gene knockin plasmids. Top: All-in-one clone expressing both CRISPR-Cas9 nuclease and one sgRNA targeted to the human AAVS1 site on chromosome 19. Botton: Donor clone for integrating a transgene of interest into the AAVS1 locus.Both plasmids are used to simultaneously co-transfect cells. Selection for puromycin resistance leads to knockin of the DNA sequences between the left homologous arm (HA-Left) and righ homologous arm (HA-Right).

 

TALEN-based Human AAVS1 Safe Harbor system

   
AAVS1_Left_TALEN-m AAVS1_Right_TALEN-m
 
 

Figure 3. Genome-TALER™ TALEN-based human AAVS1 safe harbor gene knockin plasmids. Top: Individual cllones expressing either the left- or right TALENs targeted to the human AAVS1 site on chromosome 19. Botton: Donor clone for integrating a transgene of interest into the AAVS1 locus.All 3 plasmids are used to simultaneously co-transfect cells. Selection for puromycin resistance leads to knockin of the DNA sequences between the left homologous arm (HA-Left) and righ homologous arm (HA-Right).

 

Human AAVS1 Safe Harbor validation data

(A) (B)
The_comparison_chart1-s
(C)
(D)
 

Figure 4. 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.

 

CRISPR-based Mouse ROSA26 Safe Harbor system

 
mROSA26-right-talen
     
   
 

Figure 5. Genome-CRISP™ CRISPR-based mouse ROSA26 safe harbor gene knockin plasmids. Top: All-in-one clone expressing both CRISPR-Cas9 nuclease and one sgRNA targeted to the mouse ROSA26 site on chromosome 6. Botton: Donor clone for integrating a transgene of interest into the ROSA26 locus.Both plasmids are used to simultaneously co-transfect cells. Selection for puromycin resistance leads to knockin of the DNA sequences between the left homologous arm (HA-Left) and righ homologous arm (HA-Right).

 

TALEN-based Mouse ROSA26 Safe Harbor system

   
mROSA26-left-talen mROSA26-right-talen
 
 

Figure 6. Genome-TALER™ TALEN-based mouse ROSA26 safe harbor gene knockin plasmids. Top: Individual cllones expressing either the left- or right TALENs targeted to the mouse ROSA26 site on chromosome 6. Botton: Donor clone for integrating a transgene of interest into the ROSA26 locus.All 3 plasmids are used to simultaneously co-transfect cells. Selection for puromycin resistance leads to knockin of the DNA sequences between the left homologous arm (HA-Left) and righ homologous arm (HA-Right).

 

Mouse ROSA26 Safe Harbor validation data

CRISPR-based system

(A) (B)
The_comparison_chart1-s
(C)
 

Figure 7. Mouse genome safe harbor ROSA26 gene targeting. A. ROSA26 RFP control plasmid DC-RFP-SH02 (800 ng) was co-transfected with ROSA26 sgRNA/Cas9 expression clone (600 ng) or control of only control plasmid DC-RFP-SH02 (800 ng) transfected into mouse Neuro2a 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 only ROSA26 RFP control. C. PCR primers designed to amplify the HR junctions were used to verify the specific and successful integration.

 

TALEN-based system

(A) (B)
The_comparison_chart1-s
(C)
 

Figure 8. Mouse genome safe harbor ROSA26 gene targeting A. ROSA26 RFP control plasmid DC-RFP-SH02 (800 ng) was co-transfected with ROSA26 TALEN pair (600 ng for each) or control only DC-RFP-SH02 (800 ng) transfected into mouse Neuro2a 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 only ROSA26 RFP control.C. PCR primers designed to amplify the HR junctions were used to verify the specific and successful integration.

Related Products.

Genome-CRISP™ CRISPR-Cas9 products and services – GeneCopoeia offers CRISPR-Cas9 sgRNA design and cloning services, Cas9 expression clones and other related products and services for simple and fast targeted genomic editing.

Genome-TALER™ TALEN and TALE-TF custom services – GeneCopoeia offers several levels of affordable custom services for designing, creating and validating TALENs, TALE-TFs and other TAL effector-based targeted genomic modification tools

Genome-CRISP™ CRISPR-Cas9 stable cell lines – GeneCopoeia offers stable cell lines constitutively expressing the CRISPR Cas9 nuclease for a convenient means to carry out CRISPR genome editing applications with high efficiency, especially for sgRNA validation and high throughput sgRNA library screening.

Resources

User Manual

CRISPR Human AAVS1 safe harbor User Manual

TALEN Human AAVS1 safe harbor User Manual

CRISPR Mouse ROSA26 safe harbor User Manual

TALEN Mouse ROSA26 safe harbor User Manual

Application Note

GeneCopoeia Genome Editing Tools For Safe Harbor Integration In Mice and Humans

 

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.

 

References

1. Zou, J. et al. 2009. Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells. Cell Stem Cell. 2009 Jul 2;5(1):97-110

2. Sadelain, M. et al. 2011. Safe harbours for the integration of new DNA in the human genome. Nat Rev Cancer. 2011 Dec 1;12(1):51-8.

3. van Rensburg, R. et al. 2013. Chromatin structure of two genomic sites for targeted transgene integration in induced pluripotent stem cells and hepatopoietic stem cells. Gene Therapy. 2013 20(2):201-14.

4. Papapetrou, EP. et al. 2011. Genomic safe harbors permit high ß-globin transgene expression in thalassemia induced pluripotent stem cells. Nat. Biotechnol. 2011 29(1):73-8.

5. Lombardo, A. et al. 2011. Site-specific integration and tailoring of cassette design for sustainable gene transfer. Nat. Methods. 2011 8(10):861-9