Creating highly specific nucleases by fusion of active restriction endonucleases and catalytically inactive homing endonucleases
2012 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 2, 847-860 p.Article in journal (Refereed) Published
Zinc-finger nucleases and TALE nucleases are produced by combining a specific DNA-binding module and a non-specific DNA-cleavage module, resulting in nucleases able to cleave DNA at a unique sequence. Here a new approach for creating highly specific nucleases was pursued by fusing a catalytically inactive variant of the homing endonuclease I-SceI, as DNA binding-module, to the type IIP restriction enzyme PvuII, as cleavage module. The fusion enzymes were designed to recognize a composite site comprising the recognition site of PvuII flanked by the recognition site of I-SceI. In order to reduce activity on PvuII sites lacking the flanking I-SceI sites, the enzymes were optimized so that the binding of I-SceI to its sites positions PvuII for cleavage of the composite site. This was achieved by optimization of the linker and by introducing amino acid substitutions in PvuII which decrease its activity or disturb its dimer interface. The most specific variant showed a more than 1000-fold preference for the addressed composite site over an unaddressed PvuII site. These results indicate that using a specific restriction enzyme, such as PvuII, as cleavage module, offers an alternative to the otherwise often used catalytic domain of FokI, which by itself does not contribute to the specificity of the engineered nuclease.
Place, publisher, year, edition, pages
Oxford University Press, 2012. Vol. 40, no 2, 847-860 p.
zink-finger nuclease, double-strand breaks, pvuII endonuclease, TAL effectors, targeting DNA, homologous recombination, crystal structure, cleavage domain, gene therapy, in vivo
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:umu:diva-93871DOI: 10.1093/nar/gkr788ISI: 000299095900039OAI: oai:DiVA.org:umu-93871DiVA: diva2:751561