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<p>With the increasing use of CRISPR–Cas9, detecting off-target events is essential for safety. Current methods primarily focus on guide RNA (gRNA) sequence mismatches, often overlooking the impact of DNA topology in regulating off-target activity. Here we present TOPO-seq, a high-throughput and sensitive method that identifies genome-wide off-target effects of Cas9 and base editors while accounting for DNA topology. TOPO-seq revealed that topology-induced off-target sites frequently harbor higher mismatches than the relaxed DNA sequence, with over 50% of off-target sites containing six mismatches, which are usually overlooked using previous methods. Applying TOPO-seq to three therapeutic gRNAs in hematopoietic stem cells identified 47 bona fide off-target loci, six of which are specifically induced by DNA topology. These findings highlight DNA topology as a regulator of off-target editing rates, establish TOPO-seq as a robust method for capturing DNA topology-induced off-target events and underscore its importance in off-target detection for developing safe genome-editing therapies.</p><p></p>

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TOPO-seq reveals DNA topology-induced off-target activity by Cas9 and base editors

  • 期刊论文

  • 发表日期：

    2025年4月2日

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  • Min Duan,
  • Pan Gao,
  • Yi-Zhou Zhang,
  • Yu-Long Hu,
  • Lei Zhou,
  • Zhong-Chen Xu,
  • Hou-Yuan Qiu,
  • Xiao-Han Tong,
  • Rui-Jin Ji,
  • Xin-Lin Lei,
  • Hao Yin,
  • Cun-Lan Guo,
  • Ying Zhang

摘要

With the increasing use of CRISPR–Cas9, detecting off-target events is essential for safety. Current methods primarily focus on guide RNA (gRNA) sequence mismatches, often overlooking the impact of DNA topology in regulating off-target activity. Here we present TOPO-seq, a high-throughput and sensitive method that identifies genome-wide off-target effects of Cas9 and base editors while accounting for DNA topology. TOPO-seq revealed that topology-induced off-target sites frequently harbor higher mismatches than the relaxed DNA sequence, with over 50% of off-target sites containing six mismatches, which are usually overlooked using previous methods. Applying TOPO-seq to three therapeutic gRNAs in hematopoietic stem cells identified 47 bona fide off-target loci, six of which are specifically induced by DNA topology. These findings highlight DNA topology as a regulator of off-target editing rates, establish TOPO-seq as a robust method for capturing DNA topology-induced off-target events and underscore its importance in off-target detection for developing safe genome-editing therapies.