26 June 2024 | Peng Liu, Kaushik Panda, Seth A. Edwards, Ryan Swanson, Hochul Yi, Pratheek Pandesha, Yu-Hung Hung, Gerald Klaas, Xudong Ye, Megan V. Collins, Kaiil N. Renken, Larry A. Gilbertson, Veena Veena, C. Nathan Hancock, R. Keith Slotkin
The article describes the development of a genome engineering tool that combines the natural ability of transposable elements (TEs) to insert DNA into specific sites with the programmable targeting capabilities of CRISPR-associated transposases (Cas). The authors fused the rice *Pong* transposase protein to the Cas9 or Cas12a nucleases, creating a system that can sequence-specifically target and insert custom DNA into plant genomes. This system was successfully tested in the model plant *Arabidopsis* and translated into soybean, a major global crop. The TATSI (Target-site Integration using Transposase-assisted Targeting of Sequences) system demonstrated higher frequency and accuracy of targeted integration compared to current methods, enabling the delivery of enhancers and gene cargos into individual plants. The precision of targeted insertions was confirmed through deep sequencing, showing that the majority of insertions occurred at the CRISPR cleavage site or within 4 bp of it. Off-target insertions were also investigated, and the system was shown to have a low off-target rate. The authors further demonstrated the programmability of TATSI by changing the CRISPR gRNA to target different genes and the delivery of larger cargo, including a herbicide resistance gene. The system's efficiency and precision make it a promising tool for crop genome engineering, particularly in soybean, which has been a challenging target for targeted integration due to its inefficient methods.The article describes the development of a genome engineering tool that combines the natural ability of transposable elements (TEs) to insert DNA into specific sites with the programmable targeting capabilities of CRISPR-associated transposases (Cas). The authors fused the rice *Pong* transposase protein to the Cas9 or Cas12a nucleases, creating a system that can sequence-specifically target and insert custom DNA into plant genomes. This system was successfully tested in the model plant *Arabidopsis* and translated into soybean, a major global crop. The TATSI (Target-site Integration using Transposase-assisted Targeting of Sequences) system demonstrated higher frequency and accuracy of targeted integration compared to current methods, enabling the delivery of enhancers and gene cargos into individual plants. The precision of targeted insertions was confirmed through deep sequencing, showing that the majority of insertions occurred at the CRISPR cleavage site or within 4 bp of it. Off-target insertions were also investigated, and the system was shown to have a low off-target rate. The authors further demonstrated the programmability of TATSI by changing the CRISPR gRNA to target different genes and the delivery of larger cargo, including a herbicide resistance gene. The system's efficiency and precision make it a promising tool for crop genome engineering, particularly in soybean, which has been a challenging target for targeted integration due to its inefficient methods.