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- Title
787. Engineered Fok I Heterodimers for Enhanced Zinc Finger Nuclease Specificity.
- Authors
Miller, Jeffrey C.; Holmes, Michael; Jianbin Wang; Ya-Li Lee; Rupniewski, Igor; Waite, Adam; Guschin, Dmitry; Gregory, Philip; Pabo, Carl O.; Rebar, Edward J.
- Abstract
Zinc finger nucleases have been shown to be effective tools for high-frequency genome editing with potential therapeutic applications (Urnov et al., Nature 435:646-651). Such nucleases are created by fusing the cleavage domain of the type IIs restriction enzyme Fok I to engineered zinc finger proteins. One limitation of this architecture is that the natural Fok I cleavage domain must dimerize in order to cleave DNA; therefore two different zinc finger protein-Fok I fusions are required to introduce a double-stranded break at an asymmetric target site. As a consequence, use of this technology for genome editing may expose the cell to three different nuclease species – the desired heterodimer as well as two homodimers which are irrelevant to the desired process but which may still contribute to off-target cleavage events.Creating Fok I cleavage domain variants that do not function as homodimers would eliminate the possibility of off-target cutting by these species and would also increase the availability of the desired heterodimer. In order to develop such variants, we used a process of iterative structure-based design and screening in which candidate designs were tested for their ability to cleave DNA in vitro and for their ability to potentiate gene correction in a reporter cell line. A pair of nuclease variants, each containing two mutations at the dimerization interface, were developed that worked well when paired together, but were severely impaired when paired with a second copy of themselves. These mutant cleavage domains were substituted for the wild-type FokI cleavage domains in the reagents used by Urnov et al. and tested for their ability to induce sequence alterations at the endogenous human common gamma chain gene. In this system we observed that a heterodimer of our designed cleavage domains was as active as wild type Fok I at stimulating gene correction while inducing significantly fewer overall DNA cutting events. Moreover, homodimers of the designed Fok I cleavage domain variants were completely inactive. These studies demonstrate that we have successfully developed Fok I cleavage domain variants that function as obligate heterodimers. These variants should be broadly useful for increasing the potency and specificity of designed zinc finger nucleases.Molecular Therapy (2006) 13, S305–S305; doi: 10.1016/j.ymthe.2006.08.875
- Subjects
ZINC-finger proteins; GENES; ESTERASES; CELL lines; DNA
- Publication
Molecular Therapy, 2006, Vol 13, pS305
- ISSN
1525-0016
- Publication type
Article
- DOI
10.1016/j.ymthe.2006.08.875