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Zinc-finger directed double-strand breaks within CAG repeat tracts promote repeat instability in human cells

David Mittelman1, Christopher Moye, Jason Morton

  • 1Verna and Marrs McLean Department of Biochemistry and Molecular Biology and Graduate Program in Structural and Computational Biology and Molecular Biophysics, Baylor College of Medicine, Houston, TX 77030, USA.

Proceedings of the National Academy of Sciences of the United States of America
|June 2, 2009
PubMed

Insights

Zinc finger nucleases (ZFNs) induce double-strand breaks (DSBs) in expanded CAG repeats, increasing repeat instability. DSB-induced instability is partly mediated by homology-directed repair, suggesting ZFNs can model repeat disorders.

Area of Science:

  • Molecular Biology
  • Genetics
  • Genomics

Background:

  • Expanded triplet repeats are the genetic cause of numerous neurological and skeletal disorders.
  • Understanding the mechanisms driving repeat instability is crucial for developing therapeutic strategies.

Purpose of the Study:

  • To investigate the role of double-strand break (DSB) repair in CAG x CTG repeat instability in mammalian cells.
  • To develop and utilize zinc finger nucleases (ZFNs) as tools to induce site-specific DSBs within CAG repeat sequences.

Main Methods:

  • Engineered ZFNs, comprising tandem zinc fingers fused to the FokI cleavage domain, were designed to target CAG repeat sequences.
  • In vitro cleavage assays were performed to confirm ZFN activity on CAG repeats.
  • A tissue culture assay was employed to assess CAG repeat instability following ZFN transfection in human and rodent cell lines.
  • RAD51 inhibition was used to explore the role of homology-directed repair in DSB-induced repeat instability.

Main Results:

  • ZFNs were confirmed to cleave CAG repeats in vitro.
  • Transfection of ZFN-expression vectors resulted in up to a 15-fold increase in CAG repeat alterations in cell lines.
  • Longer CAG repeats exhibited greater sensitivity to ZFN-induced cleavage.
  • Analysis of repeat alterations revealed a spectrum of events, predominantly repeat contractions, consistent with DSB repair.
  • Inhibition of RAD51 significantly reduced ZFN-induced repeat instability, implicating homology-directed repair.

Conclusions:

  • ZFNs are effective tools for inducing site-specific double-strand breaks in expanded CAG repeats.
  • DSB-induced repeat instability is mediated, in part, by homology-directed repair pathways.
  • ZFNs provide a valuable reagent for studying the impact of DSBs on CAG repeat instability and modeling associated disorders.

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