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

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