Advances in directed evolution: Mutagenesis, sensing, screening, and in silico innovation.

Directed evolution (DE) represents a pivotal interdisciplinary paradigm that synergistically integrates biotechnology, computational methodologies, and precision analytical instrumentation. It enables accelerated, controllable in vitro laboratory evolution of diverse biomolecules toward predefined functional targets. This review comprehensively summarizes fundamental principles and cutting-edge progress underlying core DE frameworks, encompassing mutagenesis engineering, biosensing, high-throughput screening (HTS), cell-free and display techniques, and integrated computational approaches. We systematically elaborate on their intrinsic features, comparative merits, inherent bottlenecks, applicable scopes, and prospective optimization trajectories. Furthermore, we emphasize the transformative capacity of these technologies for the development of organisms, enzymes, antibodies, and diverse functional biomolecules with tailored functional properties, spanning industrial, clinical, and fundamental research applications. Collectively, this work aims to provide a holistic mechanistic framework of contemporary DE platforms and inspire novel conceptual and methodological directions for future research.