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<p>Mouse intestinal organoids are ideal models for investigating intestinal development and diseases. The full potential of these models hinges on the ability to precisely engineer their genome, yet traditional methods for CRISPR-based editing in 3D cultures often surfer from low efficiency, high cytotoxicity, and inconsistent post-editing differentiation, which limits their applications. Here, we developed an electroporation approach mediated by ribonucleoprotein (RNP)-CRISPR that achieves over 90% gene editing efficiency in mouse intestinal organoids. Using this optimized method, we generated APC-knockout organoids that exhibit Wnt pathway hyperactivation, demonstrated by R-spondin1-independent growth, increased nuclear β-catenin, and enhanced proliferation. Our method addresses a critical technical gap in murine organoid research, offering a scalable platform for intestinal disease modeling.</p>

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Optimization of RNP-CRISPR for high-efficiency gene editing in mouse intestinal organoids

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年2月20日

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  • Xiya Cao,
  • Youyuan He,
  • Haozhe Guo,
  • Xiaoya Cao,
  • Dan Zhang,
  • Yuxuan Lai,
  • Weihua Yang,
  • Zixuan Ma,
  • Xinghua Yu,
  • Liren Wang,
  • Dali Li,
  • Zhiyang Zeng

摘要

Mouse intestinal organoids are ideal models for investigating intestinal development and diseases. The full potential of these models hinges on the ability to precisely engineer their genome, yet traditional methods for CRISPR-based editing in 3D cultures often surfer from low efficiency, high cytotoxicity, and inconsistent post-editing differentiation, which limits their applications. Here, we developed an electroporation approach mediated by ribonucleoprotein (RNP)-CRISPR that achieves over 90% gene editing efficiency in mouse intestinal organoids. Using this optimized method, we generated APC-knockout organoids that exhibit Wnt pathway hyperactivation, demonstrated by R-spondin1-independent growth, increased nuclear β-catenin, and enhanced proliferation. Our method addresses a critical technical gap in murine organoid research, offering a scalable platform for intestinal disease modeling.