Negative DNA supercoiling induces genome-wide Cas9 off-target activity. Newton MD et al
Description
CRISPR/Cas9 is a powerful gene-editing technology, however off-target activity remains an important consideration for therapeutic applications. We have previous-ly shown that force stretching DNA induces off-target activity and hypothesised that distortions of the DNA topology in vivo, such as negative DNA supercoiling, could reduce Cas9 specificity. Using single-molecule optical-tweezers, we demonstrate that negative supercoiling λ-DNA induces sequence-specific Cas9 off-target bind-ing at multiple sites, even at low forces. Using an adapted CIRCLE-seq approach, we detect over 10,000 negative supercoiling induced Cas9 off-target double-strand breaks genome wide, caused by increased mismatch tolerance. We further demon-strate in vivo that directed local DNA distortion increases off-target activity in cells and that induced off-target events can be detected during Cas9 genome-editing. These data demonstrate that Cas9 off-target activity is regulated by DNA topology in vitro and in vivo, suggesting that cellular processes, such as transcription and replication, could induce off-target activity at previously overlooked sites.