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<p>Detection of the off-target effects of base editors is important for identifying their safety risks but current methods for understanding their global activities have limitations in terms of sensitivity or bias by computationally selecting a subset of sites for experimental analysis. We present CHANGE-seq-BE, a method to assess the guide RNA-dependent off-target profile of both adenine and cytosine base editors that is simultaneously sensitive and unbiased. CHANGE-seq-BE relies on selective sequencing of base-editor-modified genomic DNA in vitro and provides comprehensive identification of genome-wide off-target mutations. We found that 98.8% of validated off-target sites were unique to ABE8e adenine base editors compared to Cas9 nuclease, suggesting substantially higher off-target activity of the former. We further applied CHANGE-seq-BE to support genotoxicity studies in an emergency investigational new drug application for customized adenine base editor treatment for a person with CD40L-deficient X-linked hyper IgM syndrome. Our results emphasize the importance of using a base-editor-specific method for identifying off-target activity.</p>

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Sensitive and unbiased genome-wide profiling of base-editor-induced off-target activity using CHANGE-seq-BE

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年1月2日

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  • Cicera R. Lazzarotto,
  • Varun Katta,
  • Yichao Li,
  • Garret Manquen,
  • Rachael K. Wood,
  • Jacqueline Chyr,
  • Elizabeth Urbina,
  • Azusa Matsubara,
  • GaHyun Lee,
  • Xiaolin Wu,
  • Suk See De Ravin,
  • Shengdar Q. Tsai

摘要

Detection of the off-target effects of base editors is important for identifying their safety risks but current methods for understanding their global activities have limitations in terms of sensitivity or bias by computationally selecting a subset of sites for experimental analysis. We present CHANGE-seq-BE, a method to assess the guide RNA-dependent off-target profile of both adenine and cytosine base editors that is simultaneously sensitive and unbiased. CHANGE-seq-BE relies on selective sequencing of base-editor-modified genomic DNA in vitro and provides comprehensive identification of genome-wide off-target mutations. We found that 98.8% of validated off-target sites were unique to ABE8e adenine base editors compared to Cas9 nuclease, suggesting substantially higher off-target activity of the former. We further applied CHANGE-seq-BE to support genotoxicity studies in an emergency investigational new drug application for customized adenine base editor treatment for a person with CD40L-deficient X-linked hyper IgM syndrome. Our results emphasize the importance of using a base-editor-specific method for identifying off-target activity.