TARGATT™ Genome Editing
Applied StemCell’s (ASC) proprietary TARGATT™ technology, enables fast and site-specific, stable integration of large DNA fragments into an intergenic, transcriptionally active safe harbor locus with very high efficiency. The TARGATT™ gene editing platform is versatile and can generate large fragment knock-in cell line and animal models. This technology circumvents problems associated with random integration such as position effect, and gene silencing or instability due to integration of multiple copies of the transgene.
The TARGATT™ system has been integrated into the TARGATT™ Master iPSC Line. The TARGATT™ Master iPSC Line was engineered from our well-characterized control human iPSC line, ASE-9211 (a NIST iPSC). The master cell line carries an “attP” integrase recognition landing pad in the H11 safe harbor locus. When used in conjunction with an “attB” containing donor plasmid and integrase expression, this master cell line enables site-specific integration of a transgene (up to 22kb) with very high efficiency.
In addition, the TARGATT™ site-specific knockin technology to engineer two additional TARGATT™ Master Cell Lines. The TARGATT™ Master CHO and HEK293 Cell Lines enable single-copy insertion at a safe-harbor locus (H11, ASC2). High integration efficiencies and medium to high levels of protein expression have been observed. These cell lines have the capacity to generate libraries for antibody screening. ASC can produce the library that best fits your project needs, or you can build your own library with the TARGATT™ CHO-K1 Kit (H11) or TARGATT™ HEK293 Kit (H11) for antibody library screening.
ASC is in the process of getting GLP and GMP certified and complied with. We now offer a wide range of GMP clinical-grade induced pluripotent stem cell (iPSC) related services. Applied StemCell’s GMP Grade iPSC line (Cord Blood CD34+, Male) can be engineered to express almost any gene of interest using TARGATTTM, but ASC can also conduct gene-editing services with the iPSC lines provided by customers. GMP grade knockin cell line generation service using the TARGATT™ Master iPSC Line is also available for single-copy gene insertion at a safe harbor locus.
*If you would like to work with your favorite cell line, ASC can engineer the target system into the cell line of your choice!
TARGATT™ Cell Line Advantages:
- Single copy knockin: 1 cell, 1 docking site, 1 inserted transgene
- Site-specific knockin into a high expression, safe harbor locus (H11, ASC2)
- High efficiency integration (HEK293: >40% without, >90% with drug selection; CHO: ~18% without, >90% with drug selection)
- Large cell library construction in HEK293/ CHO cells
- Cost-effective: no virus packaging time and resources
- BSL1 compatible
Applied StemCell has also used its TAGRATT™ technology to generate several TARGAT™ mouse and rat models. Custom services for TARGATT site-specific knockin mouse and rat model generation are available. As a long-standing leader in custom mouse model engineering, ASC also offers 2,000+ “off-shelf” proprietary, genetically engineered mouse models (GEMM) that are research-ready and can be directly used in gene function, drug screening, and human disease research. Applications for TARGATT™ models: Transgene overexpression models, humanized gene knockin, reporter gene knock-in, conditional knock-in, inducible expression, and Cre-driver lines.
To learn more about TARGATTTM and how you can incorporate the technology into your research, contact us today!
TARGATT CHO Master Cell Lines
Unlike other recombinases, such as Cre or Flp, the TARGATT™ integrase recognizes and recombines at two largely unrelated sites, attP and attB, in terms of their sequences. Once the integrase-mediated integration at attB and attP takes place, two new hybrid sites, attL and attR are created at the junctions. These new sites are unrecognizable by integrase; therefore, integrase reaction is uni-directional. Once the DNA is integrated, it will not be excised, making the integration process highly efficient. With TARGATT™ integrase system, the gene is integrated in exactly the Rosa26 or H11 locus permanently.
Yes. We can generate TARGATT™ Master cell lines engineered with the “attP” docking site into a safe harbor locus.
A single copy.
Any defined promoters provided by the customer or published in the literature can be used.
Your gene of interest will be specifically inserted at your choice of either of the two well-characterized loci: H11 and Rosa26 (or AAVS1 for cell lines).
To date, the largest DNA fragment we were successfully with is 22 kb. Insertion efficiency appears to decrease with increasing DNA fragment size.
Yes, TARGATT™ system is ideal for gene over-expression. Different promoters, e.g., tissue-specific promoters or ubiquitous promoters, and inducible systems (Tet On/Off, loxP-stop-loxP) can be used for tissue-specific, ubiquitous, or inducible gene expression.
Yes, you can express any reporter genes such as GFP, DsRed, mCherry, LacZ, Luciferase, and others.
Yes, the TARGATT™ system can be used to generate tissue-specific transgenic animal and cell line models. Just use a tissue-specific promoter to drive the transgene expression. Alternatively, a loxP-stop-loxP cassette can be placed between a ubiquitous promoter and the transgene. Upon crossing with tissue-specific Cre mice/ rats or treatment with Cre, the transgene will be expressed in that particular tissue.
Yes. This would be a customized service. We need to first insert the docking attP site into a desired locus using CRISPR/Cas9 and then insert the gene of interest into the attP site using TARGATT™.
Knock-in/out, Conditional Knockout Mouse / Rat Model
Custom Services Brochure
How-to Guide for TARGATT™ Transgenic Kit: Dr Ruby Chen-Tsai
Choosing the Right Genome Editing Technology for Your Mouse Models: CRISPR, TARGATT™ and Beyond...: Dr. Carlisle Landel
Advantages of TARGATT™ Technology:
- High integration efficiency (up to 40%)
- Large transgene knock-in (up to 22 kb)
- Reduced time and cost
- High level expression of the transgene
- Site-specificity allows a precise comparison of the effects of the transgenes among different lines
- Site-specific knock-in at pre-selected locus overcomes challenges associates with random integration:
- Eliminates position effect
- Integration at intergenic region ensures that no internal genes are interrupted
- Single copy gene integration eliminates repeat-induced gene silencing and genomic instability
Choosing the right genome editing technology: Applied StemCell uses two complementary genome editing technologies to generate advanced cell line and animal models very efficiently and effectively: the CRISPR/Cas9 technology and our propriety site-specific gene integration technology, TARGATT™ for large fragment (up to 22 kb) knock-in into a safe harbor locus.
(<200 Nucleotide ssODN Donor)
Knock-In Transgenes in
Safe Harbor Loci (>2kb)
(but limitations on size)
(up to 22kb)
(but limitations on size)
(2 steps: KI docking site; KI transgene)
Cre-rat resource for models of human diseases
- Zhang, H., Zheng, Q., & Chen-Tsai, R. Y. (2021). Establishment of a Cre-rat resource for creating conditional and physiological relevant models of human diseases. Transgenic research, 30(1), 91–104. https://doi.org/10.1007/s11248-020-00226-7
- Chen-Tsai, R. Y. (2020). Integrase-Mediated Targeted Transgenics Through Pronuclear Microinjection. In Transgenic Mouse (pp. 35-46). Humana, New York, NY.
- Chen-Tsai, R. Y. (2019). Using TARGATT™ Technology to Generate Site-Specific Transgenic Mice. In Microinjection (pp. 71-86). Humana Press, New York, NY.
Master Cell Line
- Chi, X., Zheng, Q., Jiang, R., Chen-Tsai, R. Y., & Kong, L. J. (2019). A system for site-specific integration of transgenes in mammalian cells. PLOS ONE, 14(7), e0219842.
Description of the technology
- Zhu, F., Gamboa, M., Farruggio, A. P., Hippenmeyer, S., Tasic, B., Schüle, B., … Calos, M. P. (2014). DICE, an efficient system for iterative genomic editing in human pluripotent stem cells. Nucleic Acids Research, 42(5), e34. http://doi.org/10.1093/nar/gkt1290.
- Tasic, B., Hippenmeyer, S., Wang, C., Gamboa, M., Zong, H., Chen-Tsai, Y., & Luo, L. (2011). Site-specific integrase-mediated transgenesis in mice via pronuclear injection. Proceedings of the National Academy of Sciences of the United States of America, 108(19), 7902–7907. http://doi.org/10.1073/pnas.1019507108.
Commentary, comparison with other transgenic methods
- Rossant, J., Nutter, L. M., & Gertsenstein, M. (2011). Engineering the embryo. Proceedings of the National Academy of Sciences, 108(19), 7659-7660.
Tet inducible mice generated by TARGATT™
- Fan, X., Petitt, M., Gamboa, M., Huang, M., Dhal, S., Druzin, M. L., … Nayak, N. R. (2012). Transient, Inducible, Placenta-Specific Gene Expression in Mice. Endocrinology, 153(11), 5637–5644. http://doi.org/10.1210/en.2012-1556.
Advantage of Hipp11 (H11) locus
- Hippenmeyer, S., Youn, Y. H., Moon, H. M., Miyamichi, K., Zong, H., Wynshaw-Boris, A., & Luo, L. (2010). Genetic Mosaic Dissection of Lis1 and Ndel1 in Neuronal Migration. Neuron, 68(4), 695–709. http://doi.org/10.1016/j.neuron.2010.09.027.
Applications for mice generated by TARGATT™ (and cited/published articles)
- Lindtner, S., Catta-Preta, R., Tian, H., Su-Feher, L., Price, J. D., Dickel, D. E., ... & Pennacchio, L. A. (2019). Genomic Resolution of DLX-Orchestrated Transcriptional Circuits Driving Development of Forebrain GABAergic Neurons. Cell reports, 28(8), 2048-2063.
- Wang, T. A., Teo, C. F., Åkerblom, M., Chen, C., Tynan-La Fontaine, M., Greiner, V. J., ... & Jan, L. Y. (2019). Thermoregulation via Temperature-Dependent PGD2 Production in Mouse Preoptic Area. Neuron, 103(2), 309-322.
- Clarke, B. A., Majumder, S., Zhu, H., Lee, Y. T., Kono, M., Li, C., ... & Byrnes, C. (2019). The Ormdl genes regulate the sphingolipid synthesis pathway to ensure proper myelination and neurologic function in mice. eLife, 8.
- Carlson, H. L., & Stadler, H. S. (2019). Development and functional characterization of a lncRNA‐HIT conditional loss of function allele. genesis, e23351.
- Chande, S., Ho, B., Fetene, J., & Bergwitz, C. (2019). Transgenic mouse model for conditional expression of influenza hemagglutinin-tagged human SLC20A1/PIT1. PloS one, 14(10), e0223052. doi:10.1371/journal.pone.0223052
- Hu, Q., Ye, Y., Chan, L. C., Li, Y., Liang, K., Lin, A., ... & Pan, Y. (2019). Oncogenic lncRNA downregulates cancer cell antigen presentation and intrinsic tumor suppression. Nature immunology, 1.
- Matharu, N., Rattanasopha, S., Tamura, S., Maliskova, L., Wang, Y., Bernard, A., ... & Ahituv, N. (2018). CRISPR-mediated activation of a promoter or enhancer rescues obesity caused by haploinsufficiency. Science, eaau0629.
- Barrett, R. D., Laurent, S., Mallarino, R., Pfeifer, S. P., Xu, C. C., Foll, M., ... & Hoekstra, H. E. (2018). The fitness consequences of genetic variation in wild populations of mice. bioRxiv, 383240.
- Ibrahim, L. A., Huang, J. J., Wang, S. Z., Kim, Y. J., Li, I., & Huizhong, W. (2018). Sparse Labeling and Neural Tracing in Brain Circuits by STARS Strategy: Revealing Morphological Development of Type II Spiral Ganglion Neurons. Cerebral Cortex, 1-14.
- Kumar, A., Dhar, S., Campanelli, G., Butt, N. A., Schallheim, J. M., Gomez, C. R., & Levenson, A. S. (2018). MTA 1 drives malignant progression and bone metastasis in prostate cancer. Molecular oncology.
- Jang, Y., Wang, C., Broun, A., Park, Y. K., Zhuang, L., Lee, J. E., ... & Ge, K. (2018). H3. 3K4M destabilizes enhancer epigenomic writers MLL3/4 and impairs adipose tissue development. bioRxiv, 301986. doi:https://doi.org/10.1101/301986
- Tang, Y., Kwon, H., Neel, B. A., Kasher-Meron, M., Pessin, J., Yamada, E., & Pessin, J. E. (2018). The fructose-2, 6-bisphosphatase TIGAR suppresses NF-κB signaling by directly inhibiting the linear ubiquitin assembly complex LUBAC. Journal of Biological Chemistry, jbc-RA118.
- Chen, M., Geoffroy, C. G., Meves, J. M., Narang, A., Li, Y., Nguyen, M. T., ... & Elzière, L. (2018). Leucine Zipper-Bearing Kinase Is a Critical Regulator of Astrocyte Reactivity in the Adult Mammalian CNS. Cell Reports, 22(13), 3587-3597.
- Kido, T., Sun, Z., & Lau, Y.-F. C. (2017). Aberrant activation of the human sex-determining gene in early embryonic development results in postnatal growth retardation and lethality in mice. Scientific Reports, 7, 4113. http://doi.org/10.1038/s41598-017-04117-6.
- Nouri, N., & Awatramani, R. (2017). A novel floor plate boundary defined by adjacent En1 and Dbx1 microdomains distinguishes midbrain dopamine and hypothalamic neurons. Development, 144(5), 916-927.
- Li, K., Wang, F., Cao, W. B., Lv, X. X., Hua, F., Cui, B., ... & Yu, J. M. (2017). TRIB3 Promotes APL Progression through Stabilization of the Oncoprotein PML-RARα and Inhibition of p53-Mediated Senescence. Cancer Cell, 31(5), 697-710.
- Matharu, N., Rattanasopha, S., Maliskova, L., Wang, Y., Hardin, A., Vaisse, C., & Ahituv, N. (2017). Promoter or Enhancer Activation by CRISPRa Rescues Haploinsufficiency Caused Obesity. bioRxiv, 140426.
- Jiang, T., Kindt, K., & Wu, D. K. (2017). Transcription factor Emx2 controls stereociliary bundle orientation of sensory hair cells. eLife, 6, e23661.
- Booze, M. L., Hansen, J. M., & Vitiello, P. F. (2016). A Novel Mouse Model for the Identification of Thioredoxin-1 Protein Interactions. Free Radical Biology & Medicine, 99, 533–543. http://doi.org/10.1016/j.freeradbiomed.2016.09.013.
- Feng, D., Dai, S., Liu, F., Ohtake, Y., Zhou, Z., Wang, H., ... & Hayat, U. (2016). Cre-inducible human CD59 mediates rapid cell ablation after intermedilysin administration. The Journal of clinical investigation, 126(6), 2321-2333.
- Sun, N., Yun, J., Liu, J., Malide, D., Liu, C., Rovira, I. I., … Finkel, T. (2015). Measuring in vivo mitophagy. Molecular Cell, 60(4), 685–696. http://doi.org/10.1016/j.molcel.2015.10.009.
- Devine, W. P., Wythe, J. D., George, M., Koshiba-Takeuchi, K., & Bruneau, B. G. (2014). Early patterning and specification of cardiac progenitors in gastrulating mesoderm. eLife, 3, e03848. http://doi.org/10.7554/eLife.03848.
- Fogg, P. C. M., Colloms, S., Rosser, S., Stark, M., & Smith, M. C. M. (2014). New Applications for Phage Integrases. Journal of Molecular Biology, 426(15), 2703–2716. http://doi.org/10.1016/j.jmb.2014.05.014.
- Chen-Tsai, R. Y., Jiang, R., Zhuang, L., Wu, J., Li, L., & Wu, J. (2014). Genome editing and animal models. Chinese science bulletin, 59(1), 1-6.
- Park, K.-E., Park, C.-H., Powell, A., Martin, J., Donovan, D. M., & Telugu, B. P. (2016). Targeted Gene Knockin in Porcine Somatic Cells Using CRISPR/Cas Ribonucleoproteins. International Journal of Molecular Sciences, 17(6), 810. http://doi.org/10.3390/ijms17060810.
- Guenther, C. A., Tasic, B., Luo, L., Bedell, M. A., & Kingsley, D. M. (2014). A molecular basis for classic blond hair color in Europeans. Nature Genetics, 46(7), 748–752. http://doi.org/10.1038/ng.2991.
- Villamizar, C. A. (2014). Characterization of the vascular pathology in the acta2 r258c mouse model and cerebrovascular characterization of the acta2 null mouse. UT GSBS Dissertations and These (Open Access). Paper 508 (2014)