Establishing a semi-homology-directed recombination method for precision gene integration in axolotls

作者：Liqun Wang, Yan Hu, Yuanhui Qiu, Huiting Lin, Xiang Li, Sulei Fu, Yan-Yun Zeng, Maria Ghouse, Cheng Long, Yanmei Liu, Ji‐Feng Fei · 发表于：Journal of genetics and genomics/Journal of Genetics and Genomics · 年份：2025 · DOI：10.1016/j.jgg.2025.03.001 · 被引用次数：1 · 研究领域：CRISPR and Genetic Engineering、Pluripotent Stem Cells Research、Developmental Biology and Gene Regulation

The axolotl is broadly used in regenerative, developmental, and evolutionary biology research. Targeted gene knock-in is crucial for precision transgenesis, enabling disease modeling, visualization, tracking, and functional manipulation of specific cells or genes of interest (GOIs). Existing CRISPR/Cas9-mediated homology-independent method for gene knock-in often causes “scars/indels” at integration junctions. Here, we develop a CRISPR/Cas9-mediated semi-homology-directed recombination (HDR) knock-in method using a donor construct containing a single homology arm for precise GOI integration. This semi-HDR approach achieves seamless single-end integration of the Cherry reporter gene and a large inducible Cre cassette into intronless genes like Sox2 and Neurod6 in axolotls , which are challenging to modify with the homology-independent method. Additionally, we integrate the inducible Cre cassette into intron-containing loci (e.g., Nkx2.2 and FoxA2 ) without introducing indels via semi-HDR. GOIs are properly expressed in F0 founders, with approximately 5%–10% showing precise integration confirmed by genotyping. Furthermore, using the Nkx2.2:CreER T2 line, we fate-map spinal cord p3 neural progenitor cells, revealing that Nkx2.2 + cells adopt different lineages in development and regeneration, preferentially generating motoneurons over oligodendrocytes during regeneration. Overall, this semi-HDR method balances efficiency and precision in GOI integration, providing a valuable too...