Newton, MD; Losito, M; Smith, QM; Parnandi, N; Taylor, BJ; Akcakaya, P; Maresca, M; ... Rueda, DS; + view all Newton, MD; Losito, M; Smith, QM; Parnandi, N; Taylor, BJ; Akcakaya, P; Maresca, M; van Eijk, P; Reed, SH; Boulton, SJ; King, GA; Cuomo, ME; Rueda, DS; - view fewer (2023) Negative DNA supercoiling induces genome-wide Cas9 off-target activity. Molecular Cell , 83 (19) 3533-3545.e5. 10.1016/j.molcel.2023.09.008.

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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.

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