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 func...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: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 the integration of GOIs, providing a valuable tool for generating knock-in axolotls and potentially extending to other species.展开更多
基金supported by the National Key R&D Program of China(2021YFA0805000,2023YFA1800600,2019YFE0106700)the National Natural Science Foundation of China(92268114,31970782,32070819)+1 种基金the High-level Hospital Construction Project of GuangdongProvincial People'sHospital(DFJHBF202103 and KJ012021012)BGI grant(BGIRSZ20210002).
文摘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: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 the integration of GOIs, providing a valuable tool for generating knock-in axolotls and potentially extending to other species.
