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

Molecular and Cellular Biochemistry 2026 Open access

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Authors: Xiya Cao, Youyuan He, Haozhe Guo, Xiaoya Cao, Dan Zhang, Yuxuan Lai, Weihua Yang, Zixuan Ma, Xinghua Yu, Liren Wang, Dali Li, Zhiyang Zeng
DOI: 10.1007/s11010-026-05492-3
Cited by: 4
References: 31

Abstract

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.

Fields of Study & Subjects

CRISPR and Genetic Engineering Virus-based gene therapy research Pluripotent Stem Cells Research

Publication details

Journal / source
Molecular and Cellular Biochemistry
Publisher
Springer Science and Business Media LLC
Volume
481
Issue
4
Pages
1817–1827
Publication date
20 February 2026
Language
EN