PEAC-seq: an unbiased in vivo CRISPR off-target detection technique

Development of PEAC-seq for unbiased off-target detection and translocation Overall design: According to the screen and the tag insertion test, we attached a 21nt cytosine-depleted template to the 3' of pegRNA. We reasoned that both the on-target and the off-target cleavage sites in the genome will incorporate the complementary sequence of the template, which could further serve as a PCR handle to enrich the unknown cleavage sites caused by Cas9. Inspired by the GUIDE-seq strategy, the genomic DNA was extracted from the cells undergone Cas9 editing and segmented by Tn5. The fragments included cleaved sites were further enriched by a pair of primers, in which one primed at the inserted PCR handle and the other primed at the Tn5 adaptor.