The study presents the development of an engineered Cas12i nuclease, Cas-SF01, which is a highly efficient genome editing tool in animals and plants. The researchers used structure-guided rational design and protein engineering to optimize the uncharacterized Cas12i3 nuclease, resulting in Cas-SF01, a variant with significantly improved gene editing activity in mammalian cells. Cas-SF01 exhibits comparable or superior editing performance compared to SpCas9 and other Cas12 nucleases. It has an expanded PAM range, effectively recognizing NTTN, NATN, and TTNV PAMs. The D876R mutation was identified to reduce off-target effects while maintaining high on-target activity, leading to the development of Cas-SF01HfH (high-fidelity Cas-SF01). The study demonstrates that Cas-SF01 has high gene editing activities in mice and plants, making it a robust platform for genome editing applications in various organisms.The study presents the development of an engineered Cas12i nuclease, Cas-SF01, which is a highly efficient genome editing tool in animals and plants. The researchers used structure-guided rational design and protein engineering to optimize the uncharacterized Cas12i3 nuclease, resulting in Cas-SF01, a variant with significantly improved gene editing activity in mammalian cells. Cas-SF01 exhibits comparable or superior editing performance compared to SpCas9 and other Cas12 nucleases. It has an expanded PAM range, effectively recognizing NTTN, NATN, and TTNV PAMs. The D876R mutation was identified to reduce off-target effects while maintaining high on-target activity, leading to the development of Cas-SF01HfH (high-fidelity Cas-SF01). The study demonstrates that Cas-SF01 has high gene editing activities in mice and plants, making it a robust platform for genome editing applications in various organisms.