Abstract
BackgroundZinc finger nucleases (ZFNs) are powerful tools for gene therapy and genetic engineering. The characterization of ZFN protein stability and the development of simple methods to improve ZFN function would facilitate the application of this promising technology. However, the factors that affect ZFN protein stability and function are not yet clear. Here, we determined the stability and half-life of two ZFN proteins and examined the effect of MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal-Hl), a proteasome inhibitor, on ZFN-mediated gene modifications.Methodology/Principal FindingsZFN proteins were expressed in 293T cells after transfection of ZFN-encoding plasmids. We studied two ZFN pairs: Z-224, which targets the CCR5 gene, and K-230, which targets a region 230 kbp upstream of CCR5. Western blotting after treatment with cycloheximide showed that the half-life of these ZFN proteins was around two hours. An immunoprecipitation assay revealed that the ZFN interacts with ubiquitin molecules and undergoes polyubiquitination in vivo. Western blotting showed that the addition of MG132, a proteasomal inhibitor, increased ZFN protein levels. Finally, a surrogate reporter assay and a T7E1 assay revealed that MG132 treatment enhanced ZFN-directed gene editing.ConclusionsTo our knowledge, this is the first study to investigate ZFN protein stability and to show that a small molecule can increase ZFN activity. Our protein stability study should lay the foundation for further improvement of ZFN technology; as a first step, the use of the small molecule MG132 can enhance the efficiency of ZFN-mediated gene editing.
Highlights
Zinc finger nucleases (ZFNs) are artificial restriction enzymes that are comprised of custom-designed zinc finger proteins and a nuclease domain derived from the FokI endonuclease [1]
Our protein stability study should lay the foundation for further improvement of ZFN technology; as a first step, the use of the small molecule MG132 can enhance the efficiency of ZFN-mediated gene editing
To investigate whether ZFNs are regulated by the ubiquitin proteasome pathway (UPP) degradative pathway, we examined ZFN protein levels in the presence or absence of the proteasomal inhibitor MG132
Summary
Zinc finger nucleases (ZFNs) are artificial restriction enzymes that are comprised of custom-designed zinc finger proteins and a nuclease domain derived from the FokI endonuclease [1]. Zinc finger proteins can be designed to bind to specific sequences of DNA, allowing ZFNs to induce double- or single-strand breaks [2,3] in specific regions of a genome [1,4]. Such ZFN-induced breaks can induce mutations in genes of interest through errorprone non-homologous end joining [1,5,6,7] or lead to the modification of genes by homologous recombination in the presence of donor DNA [8,9,10] or single-stranded oligonucleotides [11]. We determined the stability and half-life of two ZFN proteins and examined the effect of MG132 (carbobenzoxyl-leucinylleucinyl-leucinal-Hl), a proteasome inhibitor, on ZFN-mediated gene modifications
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