Negative DNA supercoiling induces genome-wide Cas9 off-target activity
Matthew D. Newton, Marialucrezia Losito, Quentin M. Smith, Nishita Parnandi, Benjamin J. Taylor, Pınar Akçakaya, Marcello Maresca, Patrick van Eijk, Simon H. Reed, Simon J. Boulton, Graeme A. King, Maria Emanuela Cuomo, David Rueda
Abstract
CRISPR-Cas9 is a powerful gene-editing technology; however, off-target activity remains an important consideration for therapeutic applications. We have previously shown that force-stretching DNA induces off-target activity and hypothesized 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 binding 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 demonstrate 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.