An engineered Cas12i nuclease that is an efficient genome editing tool in animals and plants.

The type V-I CRISPR-Cas system is becoming increasingly more attractive for genome editing. However, natural nucleases of this system often exhibit low efficiency, limiting their application. Here, we used structure-guided rational design and protein engineering to optimize an uncharacterized Cas12i nuclease, Cas12i3. As a result, we developed Cas-SF01, a Cas12i3 variant that exhibits significantly improved gene editing activity in mammalian cells. Cas-SF01 shows comparable or superior editing performance compared to SpCas9 and other Cas12 nucleases. Compared to natural Cas12i3, Cas-SF01 has an expanded PAM range and effectively recognizes NTTN and noncanonical NATN and TTVN PAMs. In addition, we identified an amino acid substitution, D876R, that markedly reduced the off-target effect while maintaining high on-target activity, leading to the development of Cas-SF01HiFi (high-fidelity Cas-SF01). Finally, we show that Cas-SF01 has high gene editing activities in mice and plants. Our results suggest that Cas-SF01 can serve as a robust gene editing platform with high efficiency and specificity for genome editing applications in various organisms.