TALE-PvuII fusion proteins--novel tools for gene targeting

PloS One
Mert YanikWolfgang Wende

Abstract

Zinc finger nucleases (ZFNs) consist of zinc fingers as DNA-binding module and the non-specific DNA-cleavage domain of the restriction endonuclease FokI as DNA-cleavage module. This architecture is also used by TALE nucleases (TALENs), in which the DNA-binding modules of the ZFNs have been replaced by DNA-binding domains based on transcription activator like effector (TALE) proteins. Both TALENs and ZFNs are programmable nucleases which rely on the dimerization of FokI to induce double-strand DNA cleavage at the target site after recognition of the target DNA by the respective DNA-binding module. TALENs seem to have an advantage over ZFNs, as the assembly of TALE proteins is easier than that of ZFNs. Here, we present evidence that variant TALENs can be produced by replacing the catalytic domain of FokI with the restriction endonuclease PvuII. These fusion proteins recognize only the composite recognition site consisting of the target site of the TALE protein and the PvuII recognition sequence (addressed site), but not isolated TALE or PvuII recognition sites (unaddressed sites), even at high excess of protein over DNA and long incubation times. In vitro, their preference for an addressed over an unaddressed site is > 34,000-fol...Continue Reading

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Citations

Jan 31, 2015·Scientific Reports·Alexandre JuilleratPhilippe Duchateau
Mar 8, 2014·The Plant Journal : for Cell and Molecular Biology·Orlando de LangeThomas Lahaye
Jun 1, 2014·Nucleic Acids Research·Alfred PingoudWolfgang Wende
Jul 12, 2014·Nucleic Acids Research·Jason M WolfsDavid R Edgell
Oct 28, 2017·Chembiochem : a European Journal of Chemical Biology·Eszter NémethBéla Gyurcsik
May 3, 2018·Nucleic Acids Research·Adam J BogdanoveBarry L Stoddard

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Methods Mentioned

BETA
column chromatography
electrophoresis
PCR
transfection
cleavage assay
flow cytometry

Software Mentioned

Instant Imager
RVD

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