Supported by the AU strategic grant, the objective of the AU interdisciplinary CRISPR gene editing network is foster collaborations and innovations in CRISPR technology and applications.
Programmable precision gene editing technologies have revolutionized both academic and industrial biotechnology with applications such as perturbing gene functions, revealing disease pathogenesis, producing animal models, and discovering drugable genes. The CRISPR-Cas gene editing technology has paved the way for more efficient, cost-effective, high-throughput, and precise modification of genes in any cell or organism. As the adaptive immune system in bacteria and archaea, the CRISPR system helps them combat infections from phages and plasmids (Mojica et al., 2005). In 2012, the type II CRISPR system, CRISPR-associated protein 9 (Cas9) and guide RNA (gRNA), a small chimeric RNA molecule that can specifically guide Cas9 to any genome locus, were engineered for programmable gene editing in bacteria (Jinek et al., 2012). Shortly after, CRISPR-Cas9 was reported for programmable and multiplexed gene editing in human cells (Cong et al., 2013; Mali et al., 2013). To date, CRISPR-Cas9 gene editing technology has been successfully applied to introduce precision genetic modifications in a broad range of organisms.
Supported by the Danish Research Council for Independent Research in 2014, we have established an independent research group which focuses on further improvements of CRISPR-Cas9 genome editing technologies and applications of the CRISPR-Cas9 technology to establish in vitromodels with the overall aim of combatting human diseases (http://dream.au.dk). For example, we have developed several novel CRISPR-Cas9-based gene editing systems to improve targeting efficiency in mammalian cells, including a surrogate reporter system for selection of highly efficient CRISPR guide RNAs (gRNA) and enrichment of gene edited cells (Zhou et al., 2016), systems for multiplexed and simultaneous modification of up to 30 loci(Lin et al., 2016; Vad-Nielsen et al., 2016), novel Cas9 proteins with better gene knockout efficiency (Lin et al., 2017), and an approach for epigenetic programming of endogenous genes in primary fibroblasts (Xiong et al., 2016). Thus, we have established a research group of excellences in genome editing technology.
Although the technical requirements for generation and functional validation of CRISPR vectors have been streamlined, efficient and safe delivery of CRISPR gene editing vectors in vivo remains one of the major challenges in translating the CRISPR-Cas9 technology for human gene therapy. Unlike in vitro gene editing in cells, of which cells with desired genetic modification can be enriched by drug selection or by fluorescent-based cell sorting, the major roadblock of in vivoCRISPR gene editing is efficiency. This efficiency is defined by two factors: 1st, the efficiency of delivering the CRISPR vectors to the right cells or tissue; 2nd, the efficiency of introducing the right genetic modification. Currently, the state-of-the-art method for in vivoCRISPR gene editing is based on viral delivery using vectors such as recombinant adeno-associated virus (rAAV) and lentivirus. However, these viral vectors also face critical barriers such as potential immunogenicity and limited payload capacity (rAAV). Non-viral or a combination of viral and non-viral gene delivery methods have been explored for efficiency in vivoCRISPR gene editing(Yin et al., 2016). Thus, we aim to join excellences from genetics, gene editing, virology, nanotechnology, animal science and clinical science to develop novel DELIVERY approaches for efficient and safe in vivoCRISPR gene editing. In this network, we will focus on the efficient delivery of CRISPR gene editing vectors into liver, eye and lymphocytes.
Furthermore, we aim to broaden and strengthen CRISPR gene editing related research in AU. To our knowledge, there are a growing number of research groups at AU using or going to apply the CRISPR gene editing technologies in their research. More importantly, more and more research scientists from our clinical department are considering the potential application of CRISPR technology in preclinical investigation or clinical gene therapy of deadly diseases. Thus, it is urgently needed to establish such an interdisciplinary network on CRISPR technology and application at Aarhus University, hereafter named as AU-iCRISPR. The AU-iCRISPR network will provide a networking platform for scientists from AU to carry out interdisciplinary CRISPR gene editing research projects, exchange resources and ideas, establish new collaboration, find common interests and ultimately formulate new research consortiums to apply for Horizon 2020 research projects. The AU-iCRISPR network will also function as a “CRISPR gene editing research support facility” to help researchers who are new to the technology and would like to apply that in their research.
Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A., et al.(2013). Multiplex genome engineering using CRISPR/Cas systems. Science339, 819-823.
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A., and Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science337, 816-821.
Lin, L., Petersen, T.S., Jensen, K.T., Bolund, L., Kuhn, R., and Luo, Y. (2017). Fusion of SpCas9 to E. coli Rec A protein enhances CRISPR-Cas9 mediated gene knockout in mammalian cells. J Biotechnol247, 42-49.
Lin, L., Vad-Nielsen, J., and Luo, Y. (2016). CRISPR-mediated multiplexed genetic manipulation. Oncotarget7, 80103-80104.
Mali, P., Yang, L., Esvelt, K.M., Aach, J., Guell, M., DiCarlo, J.E., Norville, J.E., and Church, G.M. (2013). RNA-guided human genome engineering via Cas9. Science339, 823-826.
Mojica, F.J., Diez-Villasenor, C., Garcia-Martinez, J., and Soria, E. (2005). Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol60, 174-182.
Mout, R., Ray, M., Yesilbag Tonga, G., Lee, Y.W., Tay, T., Sasaki, K., and Rotello, V.M. (2017). Direct Cytosolic Delivery of CRISPR/Cas9-Ribonucleoprotein for Efficient Gene Editing. ACS Nano11, 2452-2458.
Vad-Nielsen, J., Lin, L., Bolund, L., Nielsen, A.L., and Luo, Y. (2016). Golden Gate Assembly of CRISPR gRNA expression array for simultaneously targeting multiple genes. Cell Mol Life Sci73, 4315-4325.
Xiong, K., Zhou, Y., Hyttel, P., Bolund, L., Freude, K.K., and Luo, Y. (2016). Generation of induced pluripotent stem cells (iPSCs) stably expressing CRISPR-based synergistic activation mediator (SAM). Stem Cell Res17, 665-669.
Yin, H., Song, C.Q., Dorkin, J.R., Zhu, L.J., Li, Y., Wu, Q., Park, A., Yang, J., Suresh, S., Bizhanova, A., et al.(2016). Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nat Biotechnol34, 328-333.
Zhou, Y., Liu, Y., Hussmann, D., Brogger, P., Al-Saaidi, R.A., Tan, S., Lin, L., Petersen, T.S., Zhou, G.Q., Bross, P., et al.(2016). Enhanced genome editing in mammalian cells with a modified dual-fluorescent surrogate system. Cell Mol Life Sci73, 2543-2563.