Recombinase-Controlled Multiphase Condensates Accelerate Nucleic Acid Amplification and CRISPR-Based Diagnostics

Isothermal techniques for amplifying nucleic acids have found extensive applications in genotyping and diagnostic tests. These methods can be integrated with sequence-specific detection strategies, such as CRISPR-based detection, for optimal diagnostic accuracy. In particular, recombinase-based amplification uses proteins from the Escherichia virus T4 recombination system and operates effectively at moderate temperatures in field and point-of-care settings. Here, we discover that recombinase polymerase amplification (RPA) is controlled by liquid–liquid phase separation, where the condensate formation enhances the nucleic acid amplification process. While two protein components of RPA could act as scaffold proteins for condensate formation, we identify T4 UvsX recombinase as the key regulator orchestrating distinct core–shell arrangements of proteins within multiphase condensates, with the intrinsically disordered C-terminus of UvsX being crucial for phase separation. We develop volumetric imaging assays to visualize RPA condensates and the reaction progression in whole volumes, and begin to dissect how macroscopic properties such as size distribution and droplet count could contribute to the overall reaction efficiency. Spatial organization of proteins in condensates may create optimal conditions for amplification, and disruption of such structures may diminish the amplification efficiency, as we demonstrate for the case of reverse transcription-RPA. The insight that RPA functions as a multiphase condensate leads us to identify the UvsXD274A mutant, which has a distinct phase-separation propensity compared to the wild-type enzyme and can enhance RNA detection via RPA-coupled CRISPR-based diagnostics.