Albany 2013: Book of Abstracts

category image Albany 2013
Conversation 18
June 11-15 2013
©Adenine Press (2012)

Genome engineering nucleases derived from GIY-YIG homing endonucleases

Efficient targeted manipulation of complex genomes requires highly specific endonucleases to generate double-strand breaks at defined locations (Bibikova et al., 2003; Bogdanove and Voytas, 2011). The predominant engineered nucleases, zinc-finger nucleases (ZFNs) and TAL effector nucleases (TALENs) use the catalytic domain of FokI as the nuclease portion. This domain, however, functions as a dimer to non-specifically cleave DNA, meaning that ZFNs and TALENs must be designed in head-to-head pairs to target a desired sequence. To overcome this limitation and expand the toolbox of genome editing reagents, we used the N-terminal catalytic domain and interdomain linker of the monomeric GIY-YIG homing endonuclease I-TevI to create I-TevI-zinc-fingers (Tev-ZFEs), and I-TevI-TAL effectors (Tev-TALs) (Kleinstiver et al. 2012). We also made I-TevI fusions to LAGLIDADGs homing endonucleases (I-Tev-LHEs). All three fusions showed activity on model substrates on par with ZFNs and TALENs in yeast-based recombination assays. These proof-of-concept experiments demonstrate that the catalytic domain of GIY-YIG homing endonucleases can be targeted to relevant loci by fusing the domain to characterized DNA-binding platforms. Recent efforts have focused on improving the Tev-TAL platform by (1) understanding the spacing requirements between the nuclease cleavage site and the DNA-binding site, (2) probing the DNA-binding requirements of the I-TevI linker domain, and (3) demonstrating activity in mammalian systems.


This research has been supported by grants to D.E. from the Canadian Institutes for Health Research (MOP 977800) and the Natural Sciences and Engineering Research Council of Canada (311610-2010), and a grant from the National Science Foundation to A.B. (0820831).


    Bibikova, M, Beumer, K., Trautman, J.K., and D. Carroll (2003) Enhancing gene targeting with designed zinc finger nucleases. Science 300:764.

    Bogdanove, A.J. and D.F. Voytas (2011) TAL effectors: Customizable proteins for DNA targeting. Science 333: 1843-1846.

    Kleinstiver, B.P, Wolfs, J.M., Kolaczyk, T., Roberts, A.K., Hu, S.X., and D.R. Edgell (2012) Monomeric site-specific nucleases for genome editing. Proceedings of the National Academy of Sciences, USA 109(21):8061-8066. doi: 10.1073/pnas.1117984109. Epub 2012 May 7.

David Edgell
Benjamin Kleinstiver
Jason M. Wolfs
Li Wang*
Tomasz Kolaczyk
Brendon McDowell
Adam Bogdanove*

Department of Biochemistry
Western University
London, ON N6A3B1
* Department of Plant Pathology and Plant-Microbe Biology
Cornell University
Ithaca, NY14853

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