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.
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.
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 kitsare 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
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
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
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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
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
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
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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
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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.
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 offersstable 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.
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
