ArticleSTAR protocols2026
Protocol for Cas9 nickase-mediated induction and two-dimensional gel detection of i-loops at human telomeric and alpha satellite repeats.
Article in STAR protocols, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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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.
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