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Updated: Oct 8, 2026
Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter
Published on: March 31, 2022
Protocol for Cas9 nickase-mediated induction and two-dimensional gel detection of i-loops at human telomeric and
Elia Zanella1, Flavia Bruno1, Ylli Doksani1
1IFOM ETS - The AIRC Institute of Molecular Oncology, Via Adamello 16, 20139 Milan, Italy.
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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