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<p>More sensitive evaluation of the off-target effects of gene editing nucleases is crucial for human gene therapy. Here we report chromogenic assays designed for sensitive evaluation of gene editing activities using CRISPR/Cas9 test system. Based on beta-galactosidase alpha complementation, qualitative and quantitative evaluations of the target and off-target effects of CRISPR/Cas9 were well established through the color alteration of the E.coli colonies. In addition to target effect analysis, these new assays provide extremely sensitive and efficient tool to profile the off-target effects with one or more bases mismatched between the targets and the gRNAs. Moreover, these assays allow the identification of gene editing effects for off-targets with one base mismatched PAM sites.</p>

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Coloring target and off-target effects of genetically modified nucleases by blue & white colony assays

  • 期刊论文

  • 发表日期：

    2025年11月7日

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  • Xumeng Chen,
  • Li Xiao,
  • Qian Wang,
  • Lin Zhou,
  • Yingying Xu,
  • Cuilan Zhou,
  • Minhui Dai,
  • Fengjiao Wang,
  • Huifen Xu,
  • Dixian Luo,
  • Pierre Sirois,
  • Kai Li,
  • Duanfang Liao,
  • Jia Zhang

摘要

More sensitive evaluation of the off-target effects of gene editing nucleases is crucial for human gene therapy. Here we report chromogenic assays designed for sensitive evaluation of gene editing activities using CRISPR/Cas9 test system. Based on beta-galactosidase alpha complementation, qualitative and quantitative evaluations of the target and off-target effects of CRISPR/Cas9 were well established through the color alteration of the E.coli colonies. In addition to target effect analysis, these new assays provide extremely sensitive and efficient tool to profile the off-target effects with one or more bases mismatched between the targets and the gRNAs. Moreover, these assays allow the identification of gene editing effects for off-targets with one base mismatched PAM sites.