A Programmable Eukaryotic Argonaute Nuclease with Dual DNA and RNA Cleavage Activity from Thermophilic Fungus Thermochaetoides thermophila

Eukaryotic Argonautes (eAgos) are well‐known for their role in utilizing RNA guides to recognize and cleave RNA targets, thereby participating in posttranscriptional regulation and RNA interference. However, it remains unclear whether certain eAgos can also cleave DNA at mesophilic temperatures. Here, we describe a unique eAgo nuclease, TtpAgo, from the thermophilic fungus Thermochaetoides thermophila DSM 1495 that can be programmed with either DNA or RNA guides and can cleave both DNA and RNA targets at mesophilic temperatures in vitro. TtpAgo binds 12–30 nt long 5′phosphorylated or 5′hydroxylated guide molecules without strict sequence specificity and functions across a wide temperature range. Mismatches between guide and target sequences greatly impact cleavage efficiency and precision, influenced by mismatch position and nucleic acid characteristics. TtpAgo performs programmable cleavage of highly structured RNA at 37°C and 55°C. These properties of TtpAgo redefine the catalytic scope of eAgos and provide a versatile tool for manipulating nucleic acids in vitro.