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Volume 52 Issue 7
Jul.  2025
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Article Navigation >  Journal of Genetics and Genomics  > 2025  >  52(7): 942-953

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

doi: 10.1016/j.jgg.2025.03.001
  •  

    a. Key Laboratory of Brain, Cognition and Education Sciences, Ministry of Education, Institute for Brain Research and Rehabilitation, and Guangdong Key Laboratory of Mental Health and Cognitive Science, South China Normal University, Guangzhou, Guangdong 510631, China;

  •  

    b. Department of Pathology, Guangdong Provincial People's Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, Guangzhou, Guangdong 510080, China;

  •  

    c. School of Life Sciences, South China Normal University, Guangzhou, Guangdong 510631, China;

  •  

    d. The Innovation Centre of Ministry of Education for Development and Diseases, School of Medicine, South China University of Technology, Guangzhou, Guangdong 510006, China;

  •  

    e. School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong 510515, China

Funds:

This work was supported by the National Key R&D Program of China (2021YFA0805000, 2023YFA1800600, 2019YFE0106700), the National Natural Science Foundation of China (92268114, 31970782, 32070819), the High-level Hospital Construction Project of Guangdong Provincial People's Hospital (DFJHBF202103 and KJ012021012), and BGI grant (BGIRSZ20210002).

  • Received Date: 2024-09-30
  • Accepted Date: 2025-03-02
  • Rev Recd Date: 2025-03-02
    • Available Online: 2025-07-11
  • Publish Date: 2025-03-07
    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.
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