Systematic <i>trans</i>‐Activity Comparison of Several Reported Cas12f Nucleases^†

Comprehensive Summary Cas12f possesses both cis ‐ and trans ‐cleavage activities, with the former being extensively studied for its application in genome editing, while the latter remains less explored, particularly for diagnostic purposes, and is mostly focused on Un1Cas12f1. In this study, we conducted a comprehensive comparison of the trans ‐cleavage activities of four characterized Cas12f proteins, demonstrating that all four exhibit trans ‐DNase activity triggered by double‐stranded DNA (dsDNA), single‐stranded DNA (ssDNA), and single‐stranded RNA (ssRNA). Additionally, we identified distinct base preferences for trans ‐cleavage substrates among these proteins. Our further investigation into the activities of Cas12f revealed the intricate relationship between cis ‐ and trans ‐cleavage activities under various conditions. Our study provides a multifaceted characterization of the trans ‐cleavage features of Cas12f nucleases, offering new avenues for a deeper comprehension of the mechanisms underlying Cas12f's functionality.