The Francis Crick Institute
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Negative DNA supercoiling induces genome-wide Cas9 off-target activity.

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journal contribution
posted on 2023-10-09, 10:43 authored by Matthew D Newton, Marialucrezia Losito, Quentin M Smith, Nishita Parnandi, Benjamin J Taylor, Pinar Akcakaya, Marcello Maresca, Patrick van Eijk, Simon H Reed, Simon J Boulton, Graeme A King, Maria Emanuela Cuomo, David S Rueda
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.


Crick (Grant ID: CC2057, Grant title: Boulton CC2057) European Research Council (Grant ID: 742437 - TelMetab, Grant title: ERC 742437 - TelMetab)