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Peer-reviewedCC BYSTAR protocols

Protocol for Cas9 nickase-mediated induction and two-dimensional gel detection of i-loops at human telomeric and alpha satellite repeats.

  • Elia Zanella1
  • Flavia Bruno2
  • Ylli Doksani3
  1. 1IFOM ETS - The AIRC Institute of Molecular Oncology, Via Adamello 16, 20139 Milan, Italy. Electronic address: [email protected].
  2. 2IFOM ETS - The AIRC Institute of Molecular Oncology, Via Adamello 16, 20139 Milan, Italy.
  3. 3IFOM ETS - The AIRC Institute of Molecular Oncology, Via Adamello 16, 20139 Milan, Italy. Electronic address: [email protected].
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Abstract

Single-stranded DNA breaks (SSBs) at repetitive elements promote internal loops (i-loops), precursors of extrachromosomal circular DNA (eccDNA). Here, we present a protocol for inducing targeted breaks at human telomeric or alpha-satellite repeats using doxycycline-inducible Cas9 nickase variants and repeat-specific sgRNAs. We describe steps for verifying strand-specific nicking by alkaline-gel Southern blotting and detecting i-loop accumulation by two-dimensional agarose gel electrophoresis (2D gel). In this protocol, DNase I treatment of isolated nuclei is employed as a Cas9-independent control. For complete details on the use and execution of this protocol, please refer to Zanella et al.¹.

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Cite

BibTeXRIS
BibTeX
@article{zanella2026protocol,
  title = {{Protocol for Cas9 nickase-mediated induction and two-dimensional gel detection of i-loops at human telomeric and alpha satellite repeats.}},
  author = {Elia Zanella and Flavia Bruno and Ylli Doksani},
  journal = {STAR protocols},
  year = {2026},
  month = oct,
  doi = {10.1016/j.xpro.2026.104888},
  url = {https://europepmc.org/article/MED/42837263},
  note = {CC BY},
}
RIS
TY  - JOUR
TI  - Protocol for Cas9 nickase-mediated induction and two-dimensional gel detection of i-loops at human telomeric and alpha satellite repeats.
AU  - Elia Zanella
AU  - Flavia Bruno
AU  - Ylli Doksani
PY  - 2026
DA  - 2026/10/06
JO  - STAR protocols
DO  - 10.1016/j.xpro.2026.104888
UR  - https://europepmc.org/article/MED/42837263
AB  - Single-stranded DNA breaks (SSBs) at repetitive elements promote internal loops (i-loops), precursors of extrachromosomal circular DNA (eccDNA). Here, we present a protocol for inducing targeted breaks at human telomeric or alpha-satellite repeats using doxycycline-inducible Cas9 nickase variants and repeat-specific sgRNAs. We describe steps for verifying strand-specific nicking by alkaline-gel Southern blotting and detecting i-loop accumulation by two-dimensional agarose gel electrophoresis (2D gel). In this protocol, DNase I treatment of isolated nuclei is employed as a Cas9-independent control. For complete details on the use and execution of this protocol, please refer to Zanella et al.¹.
SN  - 2666-1667
ER  -