Liqun Wang,
Yan Hu,
Yuanhui Qiu,
Huiting Lin,
Xiang Li,
Sulei Fu,
Yan-Yun Zeng,
Maria Ghouse,
Cheng Long,
Yanmei Liu,
Ji-Feng Fei
2025, 52(7): 942-953.
doi: 10.1016/j.jgg.2025.03.001
Abstract:
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 the precise integration of GOIs. 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:CreERT2 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 the integration of GOIs, providing a valuable tool for generating knock-in axolotls and potentially extending to other species.