An Engineered PfAgo with Wide Catalytic Temperature Range and Substrate Spectrum

PfAgo, a thermophilic Argonaute nuclease from Pyrococcus furiosus , is widely used in various fields due to its high DNA‐guided DNA cleavage activity. However, its high‐temperature‐dependent cleavage activity largely restricts its applications in moderate‐temperature scenarios. In this study, PfAgo is engineered for cold adaptation based on its ternary complex structure and the attributes of cold‐adapted enzymes, yielding a series of variants with better performance at moderate temperatures. Among those, mPfAgo (K617G, L618G) exhibits significantly promoted cleavage activity at 37 °C and a wider catalytic temperature range of 30–95 °C. Its high‐temperature cleavage activity is also greatly improved, enabling its application in DNA detection with attomolar sensitivity in the presence of Mg 2+ . Additionally, mPfAgo shows versatile cleavage activities, including DNA cleavage guided by 5′OH‐gDNA, 5′P‐gDNA, or 5′COOH‐gDNA, as well as RNA cleavage with 5′OH‐gDNA, 5′P‐gDNA, 5′P‐gRNA, or 5′COOH‐gDNA as guides. Further analysis through far‐UV CD spectra and DSF indicates that mPfAgo has a more flexible structure than wild‐type PfAgo. Furthermore, this established strategy is applied to engineer TtdAgo, likewise obtaining its variants with enhanced moderate‐temperature activity and expanded substrate spectrum. In summary, this work provides a novel method for the rational design of thermophilic Agos, thereby greatly expanding their application scopes.