Fast analysis and engineering of protein function by microbe-independent deep assembly and screening

Traditional methods for engineering and sequence-fitness analysis of proteins in mammalian cells are limited by the time, cost, and labor associated with plasmid cloning and preparation. Here we present Microbe-Independent Deep Assembly and Screening (MIDAS), a deterministic platform for rapid protein variant expression and characterization in mammalian cells that bypasses microbial cloning by directly transfecting PCR-assembled genes. MIDAS enables high-quality sequence-fitness assessment of arbitrary mutational spaces, including truly deep saturation mutagenesis and combinatorial variant assembly, requiring less than one workday from initial PCR to cell transfection. Using MIDAS, we engineer a high-performance acetylcholine neurotransmitter bioluminescent indicator (ACh-NeuBI), achieving stepwise improvements in responsivity through linker engineering, single-site, and multi-site mutagenesis. We also apply MIDAS to engineer improved NanoLuc luciferase variants for multiple substrates, and to characterize the structural basis of mutational tolerance and substrate specificity. Thus, MIDAS is a versatile method for rapid plasmid-free protein engineering and sequence-fitness analysis in mammalian cells, offering a practical alternative to cloning-based approaches for many protein optimization and characterization tasks.