We report a bioinformatic workflow and subsequent discovery of a new polyethylene terephthalate (PET) hydrolase, which we named MG8, from the human saliva metagenome. MG8 has robust PET plastic degradation activities under different temperature and salinity conditions, outperforming several naturally occurring and engineered hydrolases in degrading PET. Moreover, we genetically encoded 2,3‐diaminopropionic acid (DAP) in place of the catalytic serine residue of MG8, thereby converting a PET hydrolase into a covalent binder for bio‐functionalization of PET. We show that MG8(DAP), in conjunction with a split green fluorescent protein system, can be used to attach protein cargos to PET as well as other polyester plastics. The discovery of a highly active PET hydrolase from the human metagenome—currently an underexplored resource for industrial enzyme discovery—as well as the repurposing of such an enzyme into a plastic functionalization tool, should facilitate ongoing efforts to degrade and maximize reusability of PET.

Point-of-care (POC) nucleic acid detection technologies are poised to aid gold-standard technologies in controlling the COVID-19 pandemic, yet shortcomings in the capability to perform critically needed complex detection—such as multiplexed detection for viral variant surveillance—may limit their widespread adoption. Herein, we developed a robust multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)-based detection using LwaCas13a and PsmCas13b to simultaneously diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and pinpoint the causative SARS-CoV-2 variant of concern (VOC)—including globally dominant VOCs Delta (B.1.617.2) and Omicron (B.1.1.529)—all the while maintaining high levels of accuracy upon the detection of multiple SARS-CoV-2 gene targets. The platform has several attributes suitable for POC use: premixed, freeze-dried reagents for easy use and storage; convenient direct-to-eye or smartphone-based readouts; and a one-pot variant of the multiplexed detection. To reduce reliance on proprietary reagents and enable sustainable use of such a technology in low- and middle-income countries, we locally produced and formulated our own recombinase polymerase amplification reaction and demonstrated its equivalent efficiency to commercial counterparts. Our tool—CRISPR-based detection for simultaneous COVID-19 diagnosis and variant surveillance that can be locally manufactured—may enable sustainable use of CRISPR diagnostics technologies for COVID-19 and other diseases in POC settings.

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 UvsX^D274A 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.

Polypeptides generated from proteolytic processing of protein precursors, or proteolytic proteoforms, play an important role in diverse biological functions and diseases. However, their often‐small size and intricate post‐translational biogenesis preclude the use of simple genetic tagging in their cellular studies. Herein, we develop a labeling strategy for this class of proteoforms, based on residue‐specific genetic code expansion labeling with a molecular beacon design. We demonstrate the utility of such a design by creating a molecular beacon reporter to detect amyloid‐β peptides, known to be involved in the pathogenesis of Alzheimer's disease, as they are produced from amyloid precursor protein (APP) along the endocytic pathway of living cells.